Part I Analytical Methods - Wiley-VCH I Analytical Methods ... Chaotropic lysis buffers disrupt the cell and organelle ... RNA type Symbol Basic function Prokaryotic Eukaryotic

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Part IAnalytical Methods

Epigenetic Regulation and Epigenomics: Advances in Molecular Biology and Medicine, First Edition. Edited by Robert A. Meyers.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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1RNA Methodologies

Robert E. Farrell, Jr.Penn State University, Department of Biology, 1031, Edgecomb Avenue, NY, PA17403, USA

1 Introduction 8

2 Subpopulations of RNA 92.1 Messenger RNA (mRNA) 92.2 Transfer RNA (tRNA) 102.3 Ribosomal RNA (rRNA) 102.4 Nuclear RNA 112.5 Organellar RNA 122.6 Noncoding RNA 12

3 Goals in the Purification of RNA 123.1 Goal 1: Select an Appropriate Method for Membrane Solubilization 123.2 Goal 2: Ensure Total Inhibition of Nuclease Activity 133.3 Goal 3: Remove Contaminating Proteins from the Sample 133.4 Goal 4: Concentrate the Sample 143.5 Goal 5: Select the Correct Storage Conditions for the Purified RNA 14

4 Methods of Cellular Disruption and RNA Recovery 144.1 Gentle Lysis Buffers 154.2 Harsh Lysis Buffers 154.3 Silica Separation Technology 164.4 Affinity Matrices 17

5 Inhibition of Ribonuclease Activity 175.1 Preparation of Equipment and Reagents 185.2 Inhibitors of RNase 19

6 Methods for the Analysis of RNA 20

Epigenetic Regulation and Epigenomics: Advances in Molecular Biology and Medicine, First Edition. Edited by Robert A. Meyers.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

4 RNA Methodologies

6.1 RT-PCR 206.2 Northern Analysis 236.3 Nuclease Protection Assay 256.4 Transcription Rate Assays 266.5 Dot-Blot Analysis 296.6 High-Throughput Transcription Analysis 306.7 Suppression Subtractive Hybridization (SSH) 336.8 RNAi 346.9 In Vitro Translation 36

7 Summary 37

References 37

Keywords

Chaotropic

Biologically disruptive. Chaotropic lysis buffers disrupt the cell and organelle

membranes and destroy enzymatic activity on contact.

Complementary DNA (cDNA)

DNA synthesized in vitro from an RNA template by an enzyme known as a reverse

transcriptase. cDNA can be either single- or double-stranded, and is used for RT-PCR,

nucleic acid probe synthesis, or library construction. Because cDNA can only be made

from transcripts present at the moment of cellular disruption, it is a permanent

biochemical record of the cell.

Dot-blot

A membrane-based technique for the quantification of specific RNA or DNA sequences

in a sample. The sample is usually ‘‘dot’’-configured onto a filter by vacuum

filtration through a manifold. Dot blots lack the qualitative component associated

with electrophoretic assays.

Functional genomics

Response of the genome, such as changes in gene expression, as a consequence of

experimental challenge. This most often involves the up- and downregulation of specific

genes.

RNA Methodologies 5

Heterogeneous nuclear RNA (hnRNA)

The primary product of RNA polymerase II transcription in eukaryotic cells. hnRNA

alone is processed and matured into mRNA which, in turn, is able to support the

synthesis of proteins, though some hnRNA molecules fail to mature and are degraded

in the nucleus.

Housekeeping gene

A gene that is expressed, at least theoretically, at a constant level in the cell. The

products of these genes are generally required to maintain cellular viability or

normal function. Housekeeping genes are often assayed as purportedly invariant

controls, compared to the modulation of other genes in response to experimental

challenge. Almost all known housekeeping genes show varying levels of gene

expression under specific circumstances, so there is no single all-purpose housekeeping

gene.

Hybridization

The formation of hydrogen bonds between two complementary nucleic acid molecules.

The specificity of hybridization is a direct function of the stringency of the system in

which the hybridization is being conducted.

Messenger RNA (mRNA)

The mature product of RNA polymerase II transcription. mRNA is derived from

heterogeneous nuclear RNA (hnRNA) and, in conjunction with the protein translation

apparatus, is capable of directing the synthesis of the encoded polypeptide.

Noncoding RNA (ncRNA)

A diverse population of transcripts in the cell that do not encode proteins or polypeptides.

Certain classes of noncoding RNAs have been shown to profoundly regulate the

expression of other genes.

Northern blot analysis

A technique for transferring RNA from an agarose gel matrix, after electrophoresis,

onto a filter paper for subsequent immobilization and hybridization. The information

gained from Northern blot analysis is used to assess, both qualitatively and

quantitatively, the expression of specific genes, though much more sensitive methods are

available.

6 RNA Methodologies

Nuclear runoff assayA method for labeling nascent RNA molecules in isolated nuclei. The rate at whichspecific RNAs are being transcribed can then be assayed based upon the degree of labelincorporation. See ‘‘Steady-state RNA’’ for comparison.

Nuclease protection assayA method for mapping and/or quantifying RNA transcripts. In general, hybridizationbetween probe and target RNA takes place in solution, followed by nuclease digestion(with S1 nuclease or RNase) of all molecules or parts thereof which do not actuallyparticipate in duplex formation. Nucleic acid molecules which are locked up in adouble-stranded configuration are relatively safe or protected from nuclease degradation.The undigested RNA : RNA or RNA : DNA hybrids are then precipitated and/orelectrophoresed for quantification.

Poly(A)+ tailA tract of up to 250 adenosine residues enzymatically added to the 3′ terminus ofmRNA by the nuclear enzyme poly(A) polymerase. The addition of a poly(A) tailinvolves cleavage of the primary transcript, followed by polyadenylation. Most (but notall) eukaryotic mRNAs exhibit this structure which stabilizes their 3′ terminus. Thepoly(A) tract is commonly targeted by oligo(dT) for selection of these transcripts, as wellas for priming the synthesis of first-strand cDNA.

Polymerase chain reaction (PCR)Primer-mediated, enzymatic amplification of specific cDNA or genomic DNA sequences.This technology revolutionized molecular biology in the early and mid-1990s; it is thebest known and perhaps most widely used molecular biology technique.

PrimerAn artificially synthesized, short single-stranded nucleic acid molecule that can base-pairwith a complementary sequence and which provides a free 3′-OH for any of a variety ofprimer extension-related reactions, especially PCR.

ProbeA DNA or RNA molecule which carries a label allowing it to be localized and quantifiedthroughout an experiment. Probes are used most often to hybridize to complementarysequences present among a plethora of different molecules in a nucleic acid sample, asin Northern analysis, Southern analysis, nuclease protection analyses, or DNA libraryscreening.

ProteomeThe full complement of proteins produced by a cell at a particular time. Proteomemaps are typically generated and assessed by two-dimensional electrophoresis and

RNA Methodologies 7

other techniques designed to identify, quantify, and characterize the products oftranslation.

Real-time PCRA state-of-the art method for measuring PCR product accumulation as it is producedin each cycle, rather than measuring the final product mass at the end of the reaction(end-point PCR). Real-time PCR is widely regarded as the premier quantitative molecularbiology technique and, as such, is often referred to as quantitative PCR (qPCR).

Relative abundanceThe quantity of a particular RNA transcript relative to some other transcript in the samesample, or relative to the amount of the same transcript in other experimentally relatedsamples. This determination is most often made using PCR-based analysis, though lessquantitative, non-PCR assays may also be used.

Ribonuclease (RNase)A family of resilient enzymes which rapidly degrade RNA molecules. The control ofribonuclease activity is a key consideration in all manipulations involving RNA.

Ribonucleic acid (RNA)A polymer of ribonucleoside monophosphates, synthesized by an RNA polymerase.RNA is the product of transcription.

RNA interference (RNAi)A novel method by which specific mRNA transcripts can be transiently prevented fromparticipating in translation, or which are destroyed altogether through the formation ofa dsRNA molecule. RNAi is ‘‘loss-of-function’’ approach used to determine the role of aspecific gene; it is also known as post-transcriptional gene silencing.

Reverse transcription PCR (RT-PCR)The technology by which RNA molecules are converted into their complementaryDNA (cDNA) sequences by any one of several reverse transcriptases, followed by theamplification of the newly synthesized cDNA using PCR. Not to be confused withreal-time PCR, which may or may not involve the use of RNA.

Steady-state RNAThe final accumulation of RNA in the cell. For example, measurement of the prevalenceof a particular species of mRNA in a sample does not necessarily correlate with the rateof transcription or RNA degradation in the cell (see Nuclear runoff assay).

TranscriptionThe process by which RNA molecules are synthesized from a DNA template.

8 RNA Methodologies

TranscriptomeThe complete set of RNA molecules produced by a particular cell under a particular setof circumstances.

Cellular biochemistry is reflected in the abundance of cellular RNA specieswhich, inevitably, drives the phenotype of the cell. In order to understand morereadily the cellular response to experimental or environmental challenges, varioussubpopulations of RNA are harvested and characterized to gain insight to differentialexpression of genes, and possibly also the subcellular level at which these genesare modulated. RNA is isolated to answer transcription questions by measuringthe prevalence of one or more RNA species. The observed changes in transcriptabundance may then be related to morphological or physiological differences in thecells or tissues under investigation. The expedient isolation of high-quality RNA isessential to support all downstream applications, and the methods to be used aredictated by the nature of the biological source material. The RNA methodologiesare diverse, with each providing a glimpse of some aspect of gene regulation with acharacteristic level of sensitivity. Each technique has both advantages and limitations,often requiring a combination of RNA-based assays to provide a more completepicture of the upregulation and downregulation of specific genes and gene families.Data from transcription-based assays are often complemented by quantifying thecognate protein(s), the levels of which often – but not always – correlate. Mostinvestigators use RNA, rather than protein, as a parameter of gene expressionbecause RNA is often easier to isolate than proteins, and because very rare transcriptscan be detected via cDNA synthesis and PCR amplification. Presently, there is nosuch powerful amplification method for proteins.

1Introduction

The isolation and characterization of ri-bonucleic acid (RNA) from cells and tissuesamples is a central and recurrent theme inmolecular biology. In particular, the purifi-cation of chemically stable and biologicallyfunctional RNA is the starting point forthe systematic evaluation of cellular bio-chemistry by standard molecular methods,including all forms of reverse transcrip-tion polymerase chain reaction (RT-PCR),as well as time-honored methods suchas Northern analysis, nuclease protection(S1 and ribonuclease (RNase) protection

assays), nuclear runoff assay, complemen-tary DNA (cDNA) library construction, andeven dot-blot analysis. Messenger RNA(mRNA) abundance is a useful parameterof gene expression; therefore, the expe-dient recovery of RNA from a biologicalsource is a critical first step for the deriva-tion of meaningful data. Difficulties inthe purification, handling, and storage ofRNA are intrinsic to the labile chemical na-ture of these molecules. These difficultiesare further compounded by the aggressivecharacter of resilient RNases, the apparentubiquity of which is undisputed. Indeed,the novice quickly learns of the absoluterequirement for management of RNase

RNA Methodologies 9

activity at each level of RNA isolation andcharacterization. Failure to do so will al-most certainly compromise the integrity ofthe resulting RNA and its probable utilityin various downstream applications.

2Subpopulations of RNA

Prior to the onset of cellular disruption, theinvestigator must determine which RNAsubpopulation is of experimental interest.For example, the precise questions beingasked of a particular set of experimentsmay require characterization of the totalcellular RNA, the cytoplasmic RNA alone,nuclear RNA alone, poly(A)+ RNA, or even

noncoding RNA species. Transcriptionalactivity is generally assayed using oneof the methods described below, suchas Northern analysis, and the data arethen validated using another method,such as nuclease protection or RT-PCR.The variegated RNA classifications aredelineated in Table 1.

2.1Messenger RNA (mRNA)

mRNA molecules are destined to serveas templates for protein synthesis via theaction of the translation apparatus in thecell. In eukaryotes, the overwhelmingmajority of mRNA transcripts are

Tab. 1 RNA types and functions.

RNA type Symbol Basic function Prokaryotic Eukaryotic

Ribosomal RNA rRNA Forms back bone of the ribosomalsubunits

Yes Yes

Transfer RNA tRNA Transports amino acids to the ribosometo support translation

Yes Yes

Messenger RNA mRNA Template for the synthesis of proteins Yes YesHeterogeneous

nuclear RNAhnRNA Large unspliced precursor of mRNA

(pre-mRNA)No Yes

Small nuclearRNA

snRNA Facilitates splicing of hnRNA intomature, functional mRNA

No Yes

Small nucleolarRNA

snoRNA Processing of immature rRNAtranscripts in the nucleolus

No Yes

SmallcytoplasmicRNA

scRNA Facilitates protein trafficking andsecretion

Yes Yes

Micro RNA miRNA Short antisense RNAs that participate inthe regulation of gene expression

No Yes

RNase P RNA – Catalytic RNA component of theenzyme/RNA complex that processestRNA molecules

Yes No

TelomeraseRNA

– RNA component of the enzyme/RNAcomplex that repairs chromosometelomeres

No Yes

Reproduced with permission from Farrell, Jr, R.E. (2010) RNA Methodologies, 4th edn. Elsevier,Academic Press [1].

10 RNA Methodologies

characterized by the presence of a tractof adenosine nucleotides known as thepoly (A) tail, and all mRNAs so-endowedare known collectively as poly(A)+ RNA.As needed, these molecules can bepurified from previously isolated cellularRNA, cytoplasmic RNA, or directly froma whole-cell lysate by using affinitychromatography. For this, oligo(dT)12–18

linked to one of several popular matrices,including paramagnetic beads, biotin, cel-lulose beads or microcrystalline cellulose,is used to sequester those transcripts thatare polyadenylated. The perceived enrich-ment is often used to increase the ability todetect very low-abundance transcripts. It isimportant to note, however, that transcriptenrichment performed to increase sen-sitivity may actually be counterproductivein some cases, because the loss of somemRNA during the enrichment proceduremay serve only to further under-representvery low-abundance mRNA. Due inno small measure to the power of thepolymerase chain reaction (PCR), andthe clever design of the required primers,poly(A)+ selection is viewed by manyinvestigators as unnecessary for mostcontemporary applications.

Poly(A)− RNA is that subpopulationof RNA lacking the tract of adenosineresidues at the 3′ terminus; it includesa small number of mRNA molecules, anoteworthy example of which are the his-tone mRNAs. The predominant membersof this class, however, include ribosomalRNA (rRNA), transfer RNA (tRNA), andother noncoding transcripts. In instanceswhere poly(A)− mRNA might not bedetected due to exclusion from a sample,either the poly(A)− fraction or a sample oftotal RNA from the same biological sourcewill contain these naturally nonadenylatedtranscripts for assay, assuming theirrespective genes are transcriptionally

active. Moreover, the depletion of poly(A)+

mRNA from a sample renders the result-ing poly(A)− fraction an excellent negativecontrol in the assay of poly(A)+ mRNAspecies. For all of these reasons, it shouldbe noted that the terms ‘‘poly(A)+ mRNA’’and ‘‘mRNA’’ are not always synonymous.Finally, mRNAs in eukaryotic cells exhibitan unusual 5′ → 5′ linkage between thefirst two nucleotides, known as the 5′

cap. This structure not only stabilizesthe 5′ end of the transcript but it alsoefficiently identifies mRNAs as candidatesfor translation, as these caps are found onmRNAs only, and not on other types oftranscripts.

2.2Transfer RNA (tRNA)

tRNA transcripts are small (74–95 nt)molecules with the responsibility of shut-tling amino acids from the cytosol to theaminoacyl site of the ribosome, in or-der to support the process of translation.These tRNAs are not consumed duringthis process but are simply returned tothe cytosol in order to acquire and trans-port additional amino acid molecules.The cognate amino acid that specifictRNA species will transport is encodedin its anticodon. Although tRNAs aresingle-stranded molecules, they fold intoa characteristic three-dimensional (3-D)clover-leaf shape, and are immediately rec-ognizable.

2.3Ribosomal RNA (rRNA)

rRNA transcripts form the backbones ofthe large and small ribosomal subunits.Depending on the organism, as many as 80or more proteins ‘‘decorate’’ the rRNAs inorder to form functional protein-synthesis

RNA Methodologies 11

Tab. 2 Comparison of the traditional Northernanalysis, nuclease protection assay, nuclear runoffassay, and RT-PCR.

Cell type Major rRNA species Electrophoretic mobility Subunit sizes Intact ribosome

Prokaryote 16S, 23S 1.5 kb, 2.9 kb 30S, 50S 70SEukaryote 18S, 28S 1.9 kb, 4.7 kb 40S, 60S 80S

factories. In prokaryotes, the small andlarge ribosomal subunits are known asthe 30S and 50S, respectively, and theireukaryotic counterparts are known as the40S subunits and 60S subunits, where Srepresents the ‘‘Svedberg unit,’’ which is asedimentation coefficient.

In the cell, the ribosome subunits aredissociated until just prior to the initiationof translation but, upon the completion oftranslation the ribosome again separatesinto its constituent subunits. rRNA isthe most abundant type of transcript inthe cell, often contributing up to 80%of the total RNA. As such, the majorrRNAs species are useful as molecularweight standards for RNA electrophoresis,as indicated in Table 2.

2.4Nuclear RNA

Nuclear RNA is often studied in conjunc-tion with the independent characterizationof cytoplasmic RNA as a means of as-sessing the level (transcriptional versuspost-transcriptional) and the degree ofregulation of various genes. It is welldocumented that a large mass of tran-scribed RNA is degraded in the nucleus;this precursor RNA never matures intomRNA capable of supporting translationin the eukaryotic cytoplasm. By com-paring the nuclear abundance and cyto-plasmic abundance of a particular RNA,

a cause–effect relationship may be dis-cerned between an experimental manip-ulation and the regulation of gene ex-pression in that system with respect toRNA biogenesis, because heterogeneousnuclear RNA (hnRNA), produced by theaction of the enzyme RNA polymeraseII, matures into mRNA. The analysisof nuclear RNA may also be performedin order to determine the rate at whichgenes are transcribed (e.g., in the nu-clear runoff assay; see below), as op-posed to the assay of steady-state RNAlevels; these data can then be used toassess the level of regulation of geneexpression.

Small nuclear RNAs (snRNAs)represent another class of nuclear RNA.These molecules typically exist as theRNA–protein complexes, known asU1, U2, U4, U5, and U6, and areconfined to the nucleus where theyare generically referred to as smallnuclear ribonucleoproteins (snRNPs, or‘‘snurps’’). snRNPs are now known toform enormous complexes referred toas spliceosomes; these have responsibilityfor the removal of noncoding intronsequences found in hnRNA and con-comitant exon ligation during mRNAbiogenesis. Yet another class of smallnucleolar RNAs (snoRNAs) is associatedwith rRNA biogenesis in the nucleolarregion, where transcription of the rRNAgenes occurs.


2.5Organellar RNA

Both mitochondria and chloroplastshave their own circular chromosomes(mitochondrial DNA, mtDNA and chloro-plast DNA, ctDNA, respectively) whichare inherited independently of nuclearchromatin, and in a non-Mendelianmanner. These unique genomes encodeproteins that remain in the organelle,although mitochondria and chloroplastseach import proteins encoded by nucleargenes to support normal organellarfunction. In contrast to cytoplasmicmRNAs, neither mitochondrial norchloroplast mRNAs exhibit a 5′ capstructure. Most mitochondrial transcriptsexhibit a 3′ relatively short poly(A) tail,while most chloroplast mRNAs are notpolyadenylated. Mitochondrial mRNAsoften possess unusual AUA and AUUtranslation start codons, rather than AUG.These start codons are usually observedvery close to the 5′ terminus, althoughthere is considerable variation from onecell type to the next.

2.6Noncoding RNA

Noncoding RNA refers to a populationof small transcripts that do not encodeproteins but, interestingly, are often inti-mately involved in the regulation of proteinsynthesis. This RNA category includesan abundant group of small cytoplasmicRNAs (scRNAs) found in the eukary-otic cytoplasm and, technically, also thewell-known rRNA and tRNA species de-scribed above. The small cytoplasmic tran-scripts are known to exist as RNA–proteincomplexes (scRNP, or ‘‘scyrps’’), and tohave a role in regulating the synthe-sis, sorting, and secretion of proteins,

as well as possible mRNA degradation.Of greatest contemporary interest to themolecular biologist are the microRNAs(miRNAs), which function as noncodingantisense regulators of protein synthesis.The formation of double-stranded RNA(dsRNA) structures via miRNA : mRNAbase-pairing (either perfectly or with amismatch) most commonly occurs nearthe 3′ end of the cognate transcript, andis able transiently to block the translationof that mRNA, or to direct its destructionaltogether.

3Goals in the Purification of RNA

Concise and thoughtful planning priorto beginning laboratory investigations isan absolute requirement for the recoveryof high-quality RNA that is capable ofsupporting biochemical analyses. Duringthe preliminary stages, an experimentaldesign for the purification of nucleicacids must in general address five specificgoals (adapted, in part, from Ref. [1]), thesuccessful achievement of which will havea profound influence on the yield, quality,and utility of the sample.

3.1Goal 1: Select an Appropriate Method forMembrane Solubilization

The first decision to be factored into anRNA isolation strategy is based on whichpopulation of RNA or subcellular com-partment is to be studied. For example,the aim might be to determine whether anobserved modulation of gene expressionin a model system is regulated transcrip-tionally, or by certain post-transcriptionalevent(s). In such an instance, the meth-ods selected for cellular disruption and


subsequent RNA isolation must permitthe analysis of salient nuclear transcriptsindependently of those localized in thecytoplasm.

The method of cell lysis will determinethe extent of subcellular disruption in asample, and is a direct function of thelysis buffer. For example, a lysis bufferthat is used successfully with tissue cul-ture cells may be entirely inappropriatefor whole-tissue samples due to the pres-ence of a cell wall (in the case of plantsand yeast) or tenacious proteins found inthe extracellular matrix (in animal tissues).The method by which membrane solubi-lization is accomplished will also dictatewhich additional steps will be required toremove DNA and protein from the RNApreparation, and whether compartmental-ized nuclear RNA and cytoplasmic RNAspecies can be purified independently ofone another. While DNA can be purgedfrom an RNA preparation with minimalfanfare, it is not possible to determine therelative contribution of transcripts fromthe nucleus and from the cytoplasm, oncethe RNAs from these two subcellular com-partments have mingled and copurified.A particular lysis procedure must likewisedemonstrate compatibility with ensuingprotocols. The main lesson is always tothink two steps ahead: the correct methodof solubilization is dependent on the plansfor the RNA after purification, and thequestions being asked of a particular study.

3.2Goal 2: Ensure Total Inhibition of NucleaseActivity

The imperative for controlling nucleaseactivity is non-negotiable. This includespurging RNase from reagents and equip-ment (extrinsic sources of nuclease activ-ity) and controlling the RNase activity in

a cell lysate (intrinsic source of nucleaseactivity). Whilst harsh lysis buffers inhibitnuclease activity in their own right, gentlelysis buffers often require the addition ofnuclease inhibitors to safeguard the RNAduring the isolation procedure. Steps forthe inhibition or elimination of RNaseactivity must, first and foremost, demon-strate compatibility with the lysis buffer.

3.3Goal 3: Remove Contaminating Proteinsfrom the Sample

The complete removal of protein from acellular lysate is of paramount importancein the isolation of both RNA and DNA.Meticulous attention to this detail isrequired, both for accurate quantificationand precision in hybridization, ligation,or reverse transcription into cDNA. Theremoval of proteins from nucleic acidsamples may be accomplished by:

1. Protein hydrolysis with proteinase K2. Salting-out of proteins3. Solubilizing proteins in guanidinium-

based buffers4. Repeated extraction with mixtures of

phenol and chloroform5. Any combination of the above.

RNA molecules are much less frag-ile than high-molecular-weight DNA, andconsequently more aggressive methodscan be employed for the removal ofproteins, including the use of phe-nol : chloroform extraction. While depro-teinization is in itself a means of control-ling RNase activity, purified RNA sampleswill be once again susceptible to nucle-ase degradation following removal of theprotein denaturant, especially as a conse-quence of latent RNase contamination.


3.4Goal 4: Concentrate the Sample

This is the final step in nearly all RNApurification schemes. The most versatilemethod for concentrating nucleic acids isprecipitation, using various combinationsof salt and alcohol (the most commonmethod is to add sodium acetate andethanol). Nucleic acids and the salt thatdrives their precipitation form complexeswhich have a greatly reduced solubilityin high concentrations of alcohol. Unlikethe precipitation of genomic DNA, that ofRNA typically requires longer incubationperiods, often at −20 ◦C. In addition, whencentrifuging samples a greater g-forcemust be applied in order to completely re-cover an RNA precipitate for subsequentanalysis. Other concentration proceduresinclude the use of commercially availableconcentrating devices, dialysis, centrifu-gation under vacuum, and binding tosilica column matrices in high-salt. To-day, silica-based purification formats arewidely used and have all but replaced thesalt and alcohol precipitation method. Inthe column format, the purified RNA canbe eluted in as small a volume as a fewmicroliters, thereby ensuring a favorablyhigh concentration of nucleic acid that canbe used directly. Care must be taken, how-ever, when handling the RNA at this stageof purification, as it will once again besusceptible to nuclease attack when theresidual, strongly denaturing lysis buffercomponents and deproteination reagentshave been removed.

3.5Goal 5: Select the Correct StorageConditions for the Purified RNA

Because of the naturally labile charac-ter of RNA, the incorrect storage of

excellent RNA samples will often result indegradation within a relatively short time.Many proposals have been made as to thecorrect temperature, buffer, and storageform for RNA but, as a general rule, RNAis most stable as an ethanol precipitateat −80 ◦C. Large samples or RNA stocksshould be stored in convenient aliquotsin sterile Tris-EDTA buffer (10 mM Tris,pH 7.4; 0.1 mM EDTA) in order to avoidrepeated freeze–thaw cycles. Long-termstorage in water is not recommended be-cause, over time, the slightly acidic pHenvironment will favor RNA degradationby acid depurination. Moreover, it is in-cumbent upon the investigator to ensurethat added RNase inhibitors for eitherlong-term or short-term storage will notinterfere with any subsequent manipula-tions and/or reactions involving the RNA.

4Methods of Cellular Disruption and RNARecovery

As suggested above, in order to select asuitable method for cellular disruptionor ‘‘solubilization,’’ consideration must begiven as to which subpopulation of RNAis desired for study, as well as the natureof the biological material to be used (cellsgrown in tissue culture versus whole tis-sues). Beyond cell and tissue disruption,the absolute necessity for the highest pu-rity, and highest quality, RNA cannot beunderstated. RNA molecules bind a varietyof cytoplasmic and nuclear proteins, anyone of which is capable of interfering withmost downstream applications, includingPCR. Consequently, lysis buffers that ef-fectively strip away RNA-binding proteinsare strongly preferred.

The removal of protein during RNA re-covery from its biological source often


begins with an application of the lysisbuffer. In other cases, the addition ofprotein denaturants post-lysis is per-formed, particularly when organellar in-tegrity must be maintained. In either case,thorough attention to this facet of nucleicacids isolation will minimize any subse-quent purity-associated problems. Whilethe details of many lysis buffer formu-lations have been reported, they may allbe classified as being either ‘‘gentle’’ or‘‘harsh.’’

4.1Gentle Lysis Buffers

Gentle lysis buffers are used when a spe-cific subpopulation of RNA is desired (e.g.,cytoplasmic RNA alone) and nuclear in-tegrity must be maintained, as with theisolation of cytoplasmic RNA. Gentle lysisbuffers, which often are slightly hypotonic,frequently contain the nonionic detergentNP-40 (Nonidet P-40; today known asIgepal CA-630). Because osmotic lysis isthe least aggressive method of cellular dis-ruption, NP-40 lysis buffers are ideal forsolubilization of the plasma membranealone, while the inclusion of low concen-trations of magnesium helps to maintainnuclear integrity [2]. Thus, the nucleus andits contents (DNA and nuclear RNA) canbe separated from the cytosol by usingdifferential centrifugation. The resultantsupernatant will be rich in cytoplasmicRNA and proteins, with the latter be-ing easily removed by repeated extractionwith phenol : chloroform, or using oneof the above-described alternatives. If de-sired, the nuclear pellet may be processedseparately for the recovery of nuclear tran-scripts. This method of cellular disruptionis ideally suited to cells harvested fromtissue culture; unfortunately, owing to thecomplex geometry and formidable nature

of whole-tissue samples, nonionic lysisbuffers are not effective with tissue sam-ples unless they are coupled with limited,nonshearing homogenization (e.g., usinga Dounce homogenizer).

The clear advantage of this isolationstrategy is that, ultimately, the material re-covered is cytoplasmic RNA alone (mRNA,tRNA, and rRNA). A disadvantage, how-ever, is that the lysis buffer is not suffi-ciently inhibitory toward RNase. When celllysis occurs, those RNases which normallyare sequestered will be liberated, and theiractivity will greatly compromise the in-tegrity of the RNA, despite the investigatorseeking diligently to maintain its purity.At this point it may be helpful to keepthe samples on ice at all times (unless theprotocol specifically dictates otherwise); itmight also help to use reagents and tubesthat have been pre-chilled on ice beforeuse. If desired, an exogenous RNase in-hibitor such as RNasin� (Promega) can beadded to the lysis buffer. Alternatively, hn-RNA (nuclear RNA) alone can be isolatedusing this same gentle lysis buffer which,when used correctly, does not cause nu-clear breakage. This facilitates the recoveryof intact nuclei that can be washed freefrom any residual cytoplasmic transcripts.

4.2Harsh Lysis Buffers

There is probably no better way to deal withseemingly recalcitrant RNases than to dis-rupt cells in a guanidinium lysis buffer[3]. On contact, guanidinium-containingbuffers distort the tertiary folding ofRNases, which results in their inactiva-tion. Other chaotropic lysis buffers whichcontain high concentrations of ionic de-tergents, such as sodium dodecylsulfate(SDS), have also been described. The in-clusion of additional RNase inhibitors to


these lysis buffers is not necessary, andsuch procedures for RNA isolation areusually carried out at room temperature.

In the presence of chaotropic agents, or-ganelle lysis accompanies disruption of theplasma membrane. Thus, nuclear RNA,genomic DNA and mitochondrial DNAwill all be copurified with cytoplasmicRNA, such that additional steps will berequired to remove the DNA from the sam-ple. In the past, the most prevalent of thesemethods was isopycnic centrifugation [4],which involved gradient centrifugation us-ing either cesium chloride (CsCl) [5] orcesium trifluoroacetate (CsTFA) [6]. Isopy-cnic separation of the biomolecules ispossible because of their differing buoy-ant densities (DNA, 1.5–1.7 g ml−1; RNA,1.7–2.0 g ml−1; protein, 1.1–1.2 g ml−1).

The differential partitioning of DNA,RNA and protein by acid–phenol extrac-tion, which was first described by Chom-czynski and Sacchi [7], led to a dramaticchange in the way that RNA (in particular)could be purified from cells and tissues.Succinctly, the organic extraction of nu-cleic acids at acidic pH causes DNA topartition to the interphase and organicphase, while RNA remains in the aque-ous phase. This approach precludes therequirement for ultracentrifugation, andthus greatly reduces the required amountof hands-on time, to the obvious bene-fit of the investigator. The popularity of‘‘acid–phenol extraction’’ has resulted inthe development of a number of nucleicacid isolation reagents that support theunceremonious purification of RNA fromboth tissues and tissue cultured cells alike.

In order to take full advantage of thedisruptive nature of the guanidinium iso-lation procedures, whilst maintaining thesubcellular compartmentalization of RNA,one worthwhile strategy is to start the isola-tion procedure with gentle nonionic lysis,

followed by the recovery of intact nuclei,which are then lysed with guanidiniumbuffer. The purification of nuclear (or cyto-plasmic) RNA then proceeds as if workingwith intact cells. This approach is particu-larly suited to the isolation of nuclear RNAfor Northern analysis.

The principal drawback when applyingthese chaotropic methods to intact cellsis the loss of any ability to discriminatebetween cytoplasmic and nuclear RNA.There is no method by which nuclearRNA can be separated from mRNA oncemixing has occurred, although size frac-tionation may result in a partial separation.Moreover, it is unfortunate that many sea-soned investigators begin to show signsof sloppiness with respect to the controlof RNase activity when working routinelywith guanidinium buffers. Whilst it is truethat RNA is safe from nuclease degrada-tion in the presence of these agents, thepurified RNA is once again susceptible tonuclease degradation.

4.3Silica Separation Technology

One of the more important improvementsin the area of nucleic acid isolation hasbeen the development of silica filters thatare small enough to be used with a stan-dard microcentrifuge. The filters consistof glass microfibers positioned in the bot-tom of small plastic insert that fits inside astandard 1.5 ml microfuge tube. The filtersare widely available, and may be used forthe efficient purification of RNA directlyfrom biological sources. They can also beused to clean up nucleic acids after restric-tion enzyme digestion, ligation reactions,cDNA synthesis, and PCR amplifications.In general, the RNA (or DNA) is boundto silica in a high-salt, chaotropic environ-ment that is produced by diluting a nucleic


acid sample in guanidinium thiocyanate.Following a series of washes, the purifiedmaterial is eluted from the matrix undervery low-salt conditions. The main ben-efit of this procedure is that the nucleicacid purification and clean-up can be per-formed within a remarkably short time,and using small volumes.

4.4Affinity Matrices

In addition to the methods described abovefor the isolation of total cellular RNA ortotal cytoplasmic RNA, certain productsare available which capture polyadeny-lated transcripts directly. For example,many mRNA isolation kits feature tractsof oligo(dT) that have been linked cova-lently to a solid support such as cellulose,polystyrene, latex, or paramagnetic beads.The polyadenylated transcripts are thencaptured through canonical base-pairingbetween the poly(A) tail and the oligo(dT)tract in a high-salt environment. The mainbenefit associated with affinity selection isan enrichment of a nucleic acid sample infavor of mRNA by minimizing the carry-over of rRNA and tRNA; enrichment inthis manner may also increase the sen-sitivity of an assay. An older variant ofaffinity selection involved poly(A)+ mRNAbeing affinity-captured by using a columnpacked with poly(U) linked to Sepharosebeads [8]. Although still available, this pro-cess is no longer generally used becauseof a perception that is a less-efficient ma-trix, and that the quantitative recovery ofRNA from a poly(U) matrix normally re-quires the use of formamide-based elutionbuffers.

Yet another variant of the affinity ma-trix approach is designed to study nu-cleic acid–protein interactions by passinga heterogeneous protein mixture over a

column packed with either RNA or DNAoligonucleotides, in order to capture pro-teins with some level of binding affinity tothe sequences on the column. The nucleicacid is often referred to as the ‘‘bait’’, whilethe proteins that can bind to it are knownas the ‘‘prey’’. The procedure, which maybe referred to as a ‘‘pull-down’’ method,is still popular for the characterization ofRNA- or DNA-binding proteins, despitethe advent of glass or plastic arrays (alsoknown as ‘‘chips’’) that can be used forproteome profiling.

5Inhibition of Ribonuclease Activity

The difficulties associated with the iso-lation of full-length, intrinsically labileRNA are further compounded by ubiq-uitous RNase activity. The RNases are afamily of enzymes which degrade RNAmolecules through both endonucleolyticand exonucleolytic activity cleavage. Thesesmall, remarkably stable enzymes resistdenaturation under harsh conditions suchas extremes of pH and autoclaving thatwould easily destroy the activity of manyother enzymes [9]. It is incumbent uponthe investigator to ensure that both theequipment and the reagents to be usedare purged of nucleases from the onsetof an experiment. For most RNA-mindedmolecular biologists, to say that a reagentor apparatus is sterile is more than likely astatement that it is RNase-free.

The method selected for controllingthe RNase activity must, first and fore-most, demonstrate compatibility with thecell lysis procedure. Occasionally, nucle-ase inhibitors are added to gentle lysisbuffers when subcellular organelles (nu-clei especially) are to be purified intact,as in the partitioning of nuclear RNA


from cytoplasmic RNA. However, keep-ing the reagents and microfuge tubesice-cold throughout the procedure is alsoan effective means of controlling nucleaseactivity. Second, the method of nucle-ase inhibition must support the integrityof the RNA throughout the subsequentfractionation or purification steps. Third,the reagents used to inhibit the RNaseactivity must be easily removed fromthe purified RNA, so as not to interferewith any subsequent manipulations. Inall cases – and especially when character-izing a system for the first time – thecontrol of nuclease activity should beaggressive. Failure to do so is likelyto yield a useless sample of degradedRNA.

5.1Preparation of Equipment and Reagents

Rule number one when working with RNAis to wear gloves during the preparationof reagents and equipment, and espe-cially during the actual RNA extractionprocedure. Finger greases are notoriouslyrich in RNase, and are generally acceptedas the single greatest source of RNasecontamination. There should be no hes-itation in changing gloves several timesduring the course of an RNA-related exper-iment. Door knobs, micropipettors, com-puter keyboards, iPods, refrigerator doorhandles, containers in which chemicalsare packaged, and other unassuming sur-faces are all potential sources of nucleasecontamination.

With respect to laboratory consumables,any plasticware that is certified as beingtissue culture-sterile is always preferredwhen working with RNA. This includesindividually wrapped serological pipettesand conical 15 and 50 ml tubes. In anyevent, these items should be handled

only when wearing gloves. Bulk-packedpolypropylene products (e.g., microfugetubes and micropipette tips) are poten-tial sources of nuclease contamination,due mainly to their being handled anddistributed with ungloved hands from asingle bag. These consumables are bestpurchased as being certified both DNase-and RNase-free. Any plastic product orother implement that will come into con-tact with an RNA sample at any time,either directly or indirectly, and whichcan withstand autoclaving, should be sotreated and set aside exclusively for RNAstudies.

When the use of glassware is unavoid-able (as when using organic reagentssuch as phenol and chloroform), the useof individually wrapped borosilicate glasspipettes is strongly preferred. Any glass-ware that must be re-used should be setaside for RNA work, and not allowed toenter general circulation in the laboratory.Contrary to popular belief, the temper-ature and pressure generated during theautoclaving cycle are usually insufficient toeliminate all RNase activity. Fortunately,however, RNases can be destroyed quiteeffectively by baking in a dry heat oven;glassware to be used should be rinsedwith RNase-free water and then baked for3–4 h at 200 ◦C. Baking pertains to glass-ware alone; any problems regarding theheating of plastics or other materials canusually be resolved by the manufacturers’technical department. Finally, it is vitalto pay attention to the expiry dates ofall compounds and solutions in the lab-oratory. Older bottles of stock solutionsin particular serve as excellent breedinggrounds for microorganisms, which shedtheir RNase into the solution. The use ofsuch a contaminated stock solution couldlead to the obliteration of an entire RNAsample.


5.2Inhibitors of RNase

Endogenous RNase activity varies tremen-dously from one biological source to thenext, and the degree to which action mustbe taken to inhibit nuclease activity is adirect function of the cell type. Knowledgeof the extent of intrinsic nuclease activ-ity is derived from two principal sources:the salient literature, and personal expe-rience. The method of RNase inhibitionis to a great extent a function of thetype of lysis buffer. Whereas, nondena-turing, osmotic lysis buffers often includea nuclease inhibitor, strongly denatur-ing (chaotropic) lysis buffers generally donot. Such chaotropic compounds includeguanidinium thiocyanate, guanidiniumHCl, sarcosyl, SDS, 8-hydroxyquinoline,CsCl, CsTFA, and/or various formulationsof organic solvents.

RNasin� may be used to inhibit nucle-ase activity and circumvent some of theproblems commonly associated with theuse of a vanadyl ribonucleoside (VDR)complex, and is compatible with a vari-ety of in vitro reactions. RNasin� inac-tivates RNase A, RNase B, and RNaseC, but not RNase T1, S1 nuclease, norRNase from Aspergillus. Care must betaken to avoid any strongly denaturingconditions that will cause the uncouplingof RNase − RNasin� complexes and thereactivation of RNase activity. RNasin� iswidely used in reverse transcription reac-tions in order to protect the integrity ofthe template RNA prior to the synthesis offirst-strand cDNA.

At one time, a VDR was a popular addi-tion to nonionic lysis buffers which aloneare ineffective for the control of RNase. Inthe absence of a VDR, the RNase-mediatedcleavage of the phosphodiester backboneof RNA results in the transient formation

of a dicyclic transition state intermediatethat is subsequently opened by reactionwith a water molecule. In its capacity asan RNA transition state analog, the VDRcomplex forms a highly stable dicyclicspecies to which the enzyme remainsirreversibly bound. Thus, nuclease activ-ity is eliminated by locking RNase and‘‘pseudo-substrate’’ in the transition state.The VDR binds tightly to a broad spec-trum of cellular RNases, including RNaseA and RNase T1, but not to RNase H, andis compatible with a variety of cell frac-tionation methods. It is important that aVDR is used selectively, however, as eventrace carry-over quantities are sufficient toinhibit the in vitro translation of purifiedmRNA. It can also interfere with reversetranscriptase activity, thereby excluding itsuse with any RT-PCR applications. Forthis reason, the VDR is no longer usedby most molecular biologists as an RNaseinhibitor.

Diethyl pyrocarbonate (DEPC), which atone time was used widely to purge RNasefrom solutions prepared in-house, has alsofallen out of favor with molecular biolo-gists. This is due to the widespread avail-ability of certified nuclease-free reagents,including sterile H2O, from virtually allbiotech vendors. DEPC is a well-knownnonspecific inhibitor of RNase that is usedto purge reagents of nuclease activity, dueto the unreliability of autoclaving alone.Strict precautions (as indicated by themanufacturer) must be taken when us-ing DEPC, however, as it is carcinogenicand potentially explosive. Clearly, it shouldbe avoided unless there is an absolutelycompelling reason for its use.

Hydrogen peroxide (H2O2) is a powerfuloxidizing agent that can render commonlaboratory surfaces nuclease-free by soak-ing for 20–30 min, followed by rinsingwith copious amounts of water that, at


the very least, has been autoclaved. Thesoaking of glass pipettes, gel box castingtrays, electrophoresis combs, graduatedcylinders, and similar implements in a 3%H2O2 solution is a very effective and inex-pensive measure. H2O2 is readily availablein pharmacies and similar stores. It isimportant NOT to use the more concen-trated forms of H2O2 (e.g., 30% H2O2)that are commonly available from chemi-cal supply companies since, at this higherconcentration H2O2 is extremely danger-ous, perhaps causing irreparable damageto acrylic gel box components and otherequipment, as well as tissue damage to theinvestigator. Old solutions of H2O2 mustalso be avoided, as they may no longer besolutions of H2O2!

Since many RNases manage to rena-ture following removal of the denaturingreagent(s), it is prudent to maintain sepa-rate containers of chemicals and stock so-lutions for exclusive use as RNA reagents.Chemical solids should be weighed outwith an RNase-free spatula, while stocksolutions should be aliquoted into suit-able volumes; any aliquots that have beenused must be discarded. While, initially,such actions may seem excessive, theymay well preclude the accidental intro-duction of RNase and facilitate an ex-pedient recovery of high-quality RNA.All laboratories should have establishedstandard operating procedures (SOPs)in place regarding RNA-related studies,and these protocols should be followedmeticulously.

6Methods for the Analysis of RNA

The evaluation of gene expression by thehybridization of RNA is possible in avariety of formats, as is the analysis of

DNA. Methods range from the traditionalto the contemporary, with each procedurehaving an applicability under a definedset of experimental conditions, as wellas a characteristic level of sensitivity.The relative merits of four such standardmethods are listed in Table 3.

6.1RT-PCR

The PCR is a primer-mediated, enzymaticmethod for the quasi-exponentialamplification of nucleic acid sequences.This method requires any one of severalthermostable DNA polymerases, twoshort oligonucleotides acting as nucleicprimer sequences, a dNTP cocktail, andthe appropriate chemistry to support theactivity of the enzyme. The primers aredesigned to base-pair to opposite strandsof the DNA template with their respective3′-OH ends facing each other. This leadsto the amplification of that sequencewhich is framed by the 5′ ends of therespective primers through a series ofheating, cooling, and primer extensionstages, the mechanics of which arediscussed in great detail elsewhere in theEncyclopedia of Molecular Cell Biologyand Molecular Medicine (EMCBMM).

RT-PCR is a two-step process. First,high-quality RNA acts as the templatefor the synthesis of first-strand cDNAwith the enzyme reverse transcriptase.The components and mechanics of thisreaction are almost identical to any otherfirst-strand cDNA synthesis reaction, anexample being the construction of a tradi-tional cDNA library. Second, the productsof the first-strand synthesis reaction arethen amplified using the PCR. Tradition-ally, the first-strand synthesis products areadded to a second tube which providesall of the cofactors necessary to support


Tab. 3 Comparison of the traditional northernanalysis, nuclease protection assay, nuclear runoffassay, and RT-PCR.

Northern analysis Nuclease protectionassay

Nuclear runoff assay RT-PCR

Advantages Provides aqualitativecomponent toRNA analysis.

Nylon filterssupport severalrounds ofhybridizationwith differentprobes.

Is compatible withtotal, cytoplasmic,or poly(A)+ RNA.

RNA is relativelystable on filter.

Is able to assessintegrity of thesample.

Higher sensitivitythan Northernanalysis.

Requires lesshandling of RNAthan other typesof analysis.

Is tolerant ofpartiallydegraded RNA.

Solutionhybridization ismore quantitativethan filterhybridization.

Can be used forsteady-state ortranscription rateassays.

Characterizes relativerate oftranscription.

Natural geometry ofthe chromatin ismaintained.

Permitssimultaneousstudy of severalgenes.

Can be used todiscerntranscriptionalversuspost-transcriptionalgene regulationwhen used inconjunction withdata fromNorthern analysis.

Providesunparalleledsensitivity whenproperlydesigned.

Providesunparalleledresolution.

Supersedes many ofthe classicaltechniques.

Minimizes theamount ofhandling of theRNA.

Very rapidtechnique.

Favors researchproductivity

Disadvantages Is the least sensitiveassay.

Denaturants can betoxic.

Requires extensivehandling of RNA.

Is a time-consumingprocess.

Provides ampleopportunity forRNasedegradation.

Characterizes onlysteady-state RNA.

Protected fragmentis smaller thannative RNA.

Nucleases,especially S1, canbe difficult tocontrol.

Assay is moresensitive to exacthybridizationparameters thanother assays.

Double-strandedprobes cancompromisequantitativenessof the assay ifreannealingoccurs.

Nuclear isolationrequires a fairamount of skill.

Probe complexity isvery large.

Unlabeledendogenous RNAcan compete withlabeled RNAduringhybridization.

Mechanics of theassay supporttranscriptelongation, and notinitiation, duringlabeling.

Much moresensitive to theprecise reactioncomponents andconditions thanthe other assays.

Exquisitely sensitiveto contaminants,especiallygenomic DNA.

Carry-overcontaminationmust beaddressed.

Optimization can betime-consumingand costly.

Reproduced with permission from Farrell, Jr, R.E. (2010) RNA Methodologies: A Laboratory Guide forIsolation and Characterization, 4th edn, Elsevier, p. 344 [1].


the amplification of these products byPCR. A more recently developed methodfor performing RT-PCR, which is nowwidely used in clinical and diagnostic lab-oratories, requires only one enzyme ina single reaction tube format (one-tubeRT-PCR). In either case, the newly syn-thesized cDNA is amplified as would bethe DNA from any other source, pred-icated upon the availability of a set ofgene-specific primers. The PCR-amplifiedcDNA products can then be quantified, orin some other way analyzed, in order tomore fully understand some aspect of nor-mal, or abnormal, cell function. RT-PCR isadvantageous because the very labile char-acter of RNA does not favor its long-termstorage. The synthesis of cDNA provides atemplate for a DNA polymerase-mediatedamplification on an immense scale; onlythose transcribed RNAs which are puri-fied from the cell can be converted intocDNA. Different tissues – even from thesame biological source – will yield dif-ferent cDNA products, such that cDNAmay be best thought of as a permanentbiochemical record of the cell. cDNA rep-resents a means by which the molecu-lar physiology of the cell can be studiedin great detail over a period of monthsor years – much longer, and with muchgreater sensitivity, than the assay of puri-fied RNA directly.

In addition to its obvious utility for thequantification of gene expression, the judi-cious design of primers permits RT-PCRto be used to map the 5′ and 3′ endsof transcripts – a method known as therapid amplification of 5′ complementaryDNA ends (5′ RACE) [10] and the rapidamplification of 3′ complementary DNAends (3′ RACE) [11], respectively. RACEis used to detect alternative transcriptinitiation, splicing, and poly(A)+ polymer-ization sites, and to identify induced and

repressed genes under a defined set ofenvironmental conditions.

Finally, RT-PCR can be performed usingtwo different platforms, namely end-pointPCR and real-time PCR; the latter methodmay also be referred to as the quantita-tive polymerase chain reaction (qPCR).End-point PCR involves amplifying thetemplate over 25–30 cycles, with a theoret-ical amplification of 2n-fold, where n is thenumber of cycles. When all of the cycleshave been completed, the reaction tube isopened and the resulting products are ana-lyzed using agarose gel electrophoresis. Inthis case, the band intensity is associatedwith product abundance, which in turnmirrors the abundance of the starting ma-terial. Both, the mechanics of end-pointPCR and the method of detection canlimit the sensitivity of end-point PCR.For example, the intensity of two bandsrepresenting two vastly different samplesmay appear identical on electrophoresiswhen one reaction depletes the primers(the so-called ‘‘plateau effect’’) after 15 cy-cles, and the other reaction depletes theprimers after 30 cycles.

Real-time PCR is widely regarded as the‘‘gold standard’’ with respect to nucleicacid detection sensitivity. In the real-timeformat, the accumulation of product in thereaction vessel is measured at the end of ev-ery cycle – that is, in real-time. As the PCRproduct accumulates, however, there willbe a directly proportional increase in flu-orescence, due to the inclusion of fluores-cent precursors in the reaction chemistry.The fluorescence detection system permitsan extremely early detection in the ampli-fication process, while the accumulationof product is reliably exponential. Witheach passing cycle, however, inefficienciesin the reaction itself compromise the am-plification efficiency of subsequent cycles.As a consequence, the true abundance


relationships among genes and amongsamples may be distorted, or even lost alto-gether, by waiting until the end of all cyclesbefore the products are analyzed. More-over, the fact that real-time quantificationoccurs in a sealed tube that is not opened atall greatly minimizes the risk of carry-overcontamination – an unfortunate occur-rence where the product from one PCR ex-periment inadvertently becomes the tem-plate in a subsequent experiment.

It is also important to note that, follow-ing recovery from the cell, intramolecularbase-pairing that results in secondary andtertiary RNA structures is problematic.Molecules in which higher-level struc-tures form are often resistant to reversetranscription, which thereby diminishestheir ability to be quantified or otherwiseassayed. This issue is often addressed byheat denaturation in the presence of oneor more compounds that interfere with hy-drogen bonding, and is performed prior toreverse transcription. Further, performingthe first-strand cDNA synthesis reactionat elevated temperatures also helps toreduce any intramolecular base-pairing;this is possible because of the availabilityof thermostable reverse transcriptases.

6.2Northern Analysis

The quintessential method for the assay ofgene expression is a method referred to asNorthern analysis [12] (it is also known col-loquially as Northern blotting, the North-ern blot analysis, and/or RNA blot anal-ysis. Northern analysis involves the elec-trophoretic separation of RNA moleculesunder denaturing conditions, with subse-quent transfer or ‘‘blotting’’ of the sampleonto a solid filter support (the so-called ‘‘fil-ter membrane’’). The RNA on the blot isthen hybridized to an appropriately labeled

nucleic acid probe which will supportsubsequent detection by autoradiography,or by chemiluminescence. Because thesamples of RNA undergo electrophoresisprior to their hybridization, the North-ern analysis provides both quantitativeand qualitative biochemical profiles of thesample. Denaturation of the RNA prior toelectrophoresis is necessary to ensure thatthe migration of the sample through thegel occurs only with respect to molecularweight, and is not distorted by the for-mation of any secondary structure that iscommonly associated with single-strandedmolecules. Thus, the length of the tran-script(s) can be determined – a datumthat cannot be discerned using othermethods.

The objective of the Northern analysis isto quantify gene expression by detectingthe relative abundance of those mRNAsin the sample which are of immediateinterest to the investigator. Whereas, inthe Southern analysis [13] the resultingdata pertains to the structure and organi-zation of genes, data derived by Northernanalysis reflects the transcriptional activityof genes.

The principal shortcoming associatedwith Northern blot data is the limitedsensitivity of the assay. The physical ap-plication and immobilization of an RNAsample onto a filter membrane renderssome of those molecules incapable ofbase-pairing to a complementary nucleicacid probe. Neither is the Northern analy-sis intended to discern the absolute massof RNA in the cell. Rather, such data maybe measured far more accurately by us-ing solution hybridization-based methods,especially real-time PCR. Hence, data de-rived from the Northern analysis must beinterpreted in the context of the relativeabundance of a particular RNA among all


28S

a b c d

18S

Fig. 1 Assessment of RNA quality. The sharpdefinition of the 28S and 18S rRNA species inlanes a and b demonstrates the integrity of thesample. RNA in lanes c and d is also high qual-ity, although an excessive amount of RNA wasapplied to these lanes. Lanes a and b: 20 μgof total cytoplasmic RNA prepared by NP-40lysis. Lanes c and d: 25 μg of total cellular

RNA (nuclear and cytoplasmic), prepared byguanidinium–acid–phenol extraction. Note thehigher molecular weight nuclear RNA species inthe sample. Reproduced with permission fromFarrell Jr, R.E. (1993) RNA Methodologies: A Lab-oratory Guide for Isolation and Characterization,Academic Press, San Diego, CA, p. 60).

of the samples involved; hence, the methodis semi-quantitative at best.

The electrophoresis of RNA is itself animportant diagnostic tool, with a hostof information being made available re-garding the integrity and probable utilityof an RNA sample by examining a rep-resentative aliquot. RNA has a highlycharacteristic profile on a denaturing gel(Fig. 1), whereby the appearance of thepredominant species – the 28S and 18SrRNAs – being an indicator of the in-tegrity of the sample. Ideally, a very lightsmearing above, between, and just barely

below the rRNAs indicates that sample isintact and is probably capable of support-ing nucleic acid hybridization. Heaviersmearing, especially below the level ofthe 18S rRNA is quite ominous, beingindicative of partially or fully degradedRNA (Fig. 2). The complete absence ofthe rRNAs indicates a completely de-graded sample. As it is clearly desirableto ascertain the integrity of a samplebefore moving on to sophisticated and of-ten time-consuming techniques, a briefperiod of electrophoresis to assess thequality of the sample should become


1 2 3 4 5

Fig. 2 Going, going, gone . . . degraded RNA. Arepresentative aliquot from four different samplesof human fibroblast RNA was electrophoresedin a 1.2% agarose-formaldehyde gel and thenstained with ethidium bromide. The RNA molec-ular weight standard is visible in lane 1. RNAin lanes 2–5 shows increasing degrees of degra-dation, most likely due to RNase contamina-tion during the isolation procedure. Especially

noteworthy is the complete absence of the 28Sand 18S rRNA species expected in high-qualityRNA. This is an excellent example of what notto do. Reproduced from with permission fromFarrell Jr, R.E. (2010) RNA Methodologies: A Labo-ratory Guide for Isolation and Characterization, 4thedn, Elsevier, Academic Press, San Diego, CA,p. 149) [1].

a standard procedure in any molecularbiology setting.

6.3Nuclease Protection Assay

The intrinsic shortcomings of the North-ern analysis mandate a different formatfor the assay of gene expression when veryexacting quantitative data are required.In contrast to the assay format of theNorthern analysis, at the heart of an assayby nuclease protection is a high stringencyhybridization between the target and probemolecules, both of which are free-floatingin solution (solution hybridization) asopposed to having the target mRNAfixed on the filter paper (mixed-phasehybridization). The driving forces behindsolution hybridization are the randommolecular collisions, the kinetics ofwhich are related directly to the totalmass of nucleic acid in the reaction tube

(probe + target + carrier = total mass).Because of the solution hybridizationformat, all complementary nucleic acidmolecules are presumed to be capable ofhybridization. The S1 nuclease protectionassay (Fig. 3) and the RNase protectionassay (Fig. 4) are methods of greatlyenhanced sensitivity and resolution, andare universally considered to be morequantitative than Northern analysis.

The best nucleic acid probes for theseassays are substantially shorter thanthe target mRNA. Upon molecularhybridization, a short double-strandedregion is generated, while the 5′ and 3′

regions of the target molecule flankingthe double-stranded area remain single-stranded. The enzyme S1 nuclease, or acombination of RNases, is then used todigest all of the nucleic acid moleculesthat did not participate in nucleic acidhybridization. Only double-strandednucleic acid molecules are resistant to


X-ray film

Gel

Target

Total cellular RNAor poly(A)+ RNA

Radiolabeled antisenseRNA or DNA

High stringencysolution hybridization

Hybridformation

S1 nuclease digestion of allnonhybridized nucleic acids

Protected fragment

Polyacrylamide GelElectrophoresis (PAGE)

Probe

12

34

Autoradiographydirectly from the gel

Fig. 3 S1 nuclease assay for the quantifica-tion of specific RNA species. Purified RNA ishybridized in solution with a labeled probesequence to form thermodynamically sta-ble hybrid molecules. Any RNA or probemolecules that do not participate in the for-mation of hybrid molecules are digestedaway by the single-strand-specific nucleaseS1, followed by electrophoresis of the intacthybrid molecules. The size and abundance

of protected RNAs are then deduced byautoradiography, performed directly from thegel. Lane 1: undigested probe; lanes 2 and3: experimental samples; lane 4: molecularweight standards. Reproduced with permissionfrom Farrell Jr, R.E. (2010) RNA Methodologies:A Laboratory Guide for Isolation and Characteri-zation, 4th edn, Elsevier, Academic Press, SanDiego, CA, p. 323) [1].

nuclease attack. The resulting productof this assay – the so-called ‘‘protectedfragment’’ – is then resolved by elec-trophoresis. By virtue of the mechanicsof this assay, the size of the protectedfragment is expected to be similar to thesize of the probe sequence itself, which isoften substantially shorter than the nativeRNA target, and can be visualized by usingautoradiography. As a direct result of solu-tion hybridization and the digestion of allnonhybridized nucleic acid molecules, theinvestigator can expect an at least 10-foldenhancement in sensitivity, compared

to Northern analysis, particularly whenperformed using antisense RNA probes.

6.4Transcription Rate Assays

The modulation of key regulatorymolecules is an integral cellular responseto both intracellular and extracellularchallenge. One fundamental goal inthe assessment of any biological modelsystem is an elucidation of the levelof gene modulation. While potentiallevels of regulation are infinite, they are


X-ray film

Gel

Target

Total cellular RNAor poly(A)+ RNA

Radiolabeledantisense RNA

High stringencysolution hybridization

Hybridformation

RNase digestion of allnonhybridized nucleic acids

Protected fragment

Polyacrylamide GelElectrophoresis (PAGE)

Probe

Autoradiographydirectly from the gel

12

34

Fig. 4 RNase protection assay for the quan-tification of specific RNA species. PurifiedRNA is hybridized in solution with a labeledantisense probe sequence to form thermo-dynamically stable double-stranded RNAmolecules. Any RNA or probe molecules thatremain single stranded are digested by anRNase cocktail. Following electrophoresis, thesize and abundance of protected RNAs arethen deduced by autoradiography, performed

directly from the gel. Lane 1: undigestedprobe; lanes 2 and 3: experimental samples;lane 4: molecular weight standards. The gen-eral approach is identical to that for the S1nuclease assay. Reproduced with permissionfrom Farrell Jr, R.E. (2010) RNA Methodologies:A Laboratory Guide for Isolation and Characteri-zation, 4th edn, Elsevier, Academic Press, SanDiego, CA, p. 324) [1].

broadly categorized as transcriptional ordue to some post-transcriptional event.The initial characterization of thesesystems commonly involves the isolation,hybridization and subsequent detection ofspecific RNA species by RT-PCR, nucleaseprotection analysis, or even Northernanalysis. While these approaches mayprovide reliable qualitative and quanti-tative data with respect to steady-statelevels of message, RNA prepared bytotal cellular lysis does not provide infor-mation about the rate of transcription,the subcellular compartmentalization

(nuclear or cytoplasmic) of the RNA underinvestigation, or the translatability of theRNA in the cytoplasm. Knowledge of theseaspects of gene expression is necessaryto elucidate the level of gene regulation,because the half-lives among RNA speciesare variable and because the half-life ofmany mRNA species can be modifiedin response to a particular xenobioticregimen or environmental stimulus.

In order to address these questions, twobasic approaches have been employed tostudy the mechanism of transcription andthe processing of the resulting transcripts


Test sample Control sample

Isolate intact nuclei

Initiatedtranscripts

Initiatedtranscripts

Incubate with 32P-UTP

Lyse nuclei to recoverradiolabeled RNA

Normalize based onincorporated cpm

Autoradiography

Gene A

Gene B

Gene C

Gene D

Gene A

Gene B

Gene C

Gene D

Test Control

Hybridize to cold probesequences immobilized

on a filter membrane

X-ray film

Fig. 5 Nuclear runoff assay. The relative rateof transcription of all genes can be assessedby incubation of intact nuclei with an NTPcocktail containing labeled UTP. Elongated, ra-diolabeled transcripts are then hybridized tononradioactive cDNA probes immobilized ona nylon filter. On autoradiography, the inten-sity of the signal from each dot is indicative of

the degree of label incorporation, and thus therelative rate of transcription of specific genesunder a define set of experimental conditions.Reproduced with permission from Farrell Jr,R.E. (2010) RNA Methodologies: A LaboratoryGuide for Isolation and Characterization, 4thedn, Elsevier, Academic Press, San Diego, CA,p. 343) [1].

in eukaryotic cells. In one approach, therate of transcription is measured in in-tact nuclei by the incorporation of la-beled precursor nucleotides into RNAtranscripts initiated on endogenous chro-matin at the time of nuclear isolation.Elongated, labeled nuclear RNA is thenpurified for hybridization to complemen-tary, membrane-bound DNA sequences.This technique, which is known asthe nuclear runoff assay (Fig. 5), is asuperbly sensitive method for measur-ing transcription rate as a function ofcell state [14, 15], and consequently iswidely used. Because it is the RNAtranscripts, rather than the probes usedto quantify their abundance, that are

radiolabeled the basic format of this assaycan be likened to a ‘‘reverse dot-blot,’’as the probe is membrane-bound andnonradiolabeled.

The principal advantage of the nuclearrunoff assay is that labeling occurswhilst maintaining the natural geometryof the transcription apparatus. Themechanics and reaction conditions ofthe assay promote the elongation ofinitiated transcripts, but are not believedto support new initiation events. Thedegree of labeling of any particular RNAspecies, which is indicative of the relativetranscription rate of a specific gene, maythen be assessed by liquid scintillationcounting (a specific type of radioactive


detection), coupled with autoradiography.These data correlate directly with thenumber of RNA polymerase moleculesengaged in transcribing a specific gene,and indirectly with the transcriptionalefficiency of regulatory sequences associ-ated with the gene under a defined set ofexperimental conditions. When used inconjunction with a steady-state analysis ofcytoplasmic RNA species, data from thenuclear runoff assay may be used to assesswhether an observed gene modulationis a result of a change in the synthesis(transcriptional control) or a change inthe splicing/nucleocytoplasmic transport/mRNA stability (post-transcriptionalcontrol).

The nuclear runoff assay permits the si-multaneous analysis of several genes, allof which are presumably transcribed inisolated nuclei at the same relative ratesas in intact cells. The most critical pa-rameter by far is the preparation of nucleiprior to labeling. Indeed, the success of theassay is almost entirely dependent on thespeed with which nuclei are harvested andradiolabeled with the precursor, uridinetriphosphate (UTP). Failure to generatehigh-specific activity RNA is usually a di-rect result of inexperienced handling of thenuclei prior to the labeling step. The nucleiare most often isolated by incubating thecells in a nonionic, hypotonic lysis buffer,in isoosmotic sucrose buffer containingTriton X-100, or by using non-aqueousmethods [16]. The nuclear purificationmust also be carried out in such a wayas to preserve RNA polymerase activityand nuclear structure during the isolationfrom cells cultured in vitro or, if absolutelynecessary, from tissue. The harvested nu-clei, if not labeled immediately, may bestored frozen either in liquid nitrogen orat −70 ◦C in a freezer for several months,

without any significant loss of labelingpotential.

6.5Dot-Blot Analysis

The isolation of high-quality RNA fromtissue culture cells and whole-cell samplesis merely the first (albeit the most critical)step in the evaluation of a model system.Procedures such as Northern analysis, nu-clease protection analysis, and conversioninto cDNA can be a time-consuming andexpensive option, and should not im-mediately be deemed necessary. Whenevaluating a model system, cell type,or experimental regimen for the firsttime, it may be worthwhile quantify-ing the mRNAs of interest by usinga dot-blot analysis. This simple tech-nique allows definitive statements to bemade regarding the biochemical composi-tion of a sample, but without investingexcessive man-hours and laboratory re-sources.

In order to dot-blot RNA, denaturedsamples are applied directly onto a mem-brane under vacuum, using a multi-welldot-blot filtration manifold (Fig. 6). Thesamples are immobilized onto the sur-face of the membrane, followed by nucleicacid hybridization. In this way, dot-blotsand a closely related variant known asslot-blots permit the rapid detection ofthe relative amounts of a particular RNAin a sample. Salient information canbe obtained from purified RNA samplesor whole-cell lysates, without performingelectrophoresis or any form of PCR.These methods are reserved for the later,more exacting analysis of a system af-ter the preliminary information has beenderived.

Dot-blotting represents an excellentmethod for the analysis of gene expression


Fig. 6 Minifold I dot-blot apparatus. Sample di-lutions are applied under vacuum directly to thesurface of the filter membrane resting beneaththe face plate. The geometric arrangement of the

samples allows easy visual examination of severalsamples and also facilitates digital image analy-sis. Photograph courtesy of Schleicher & Schuell,Inc., Keene, NH.

when large numbers of samples areto be evaluated simultaneously, such asan experiment requiring numerous timepoints. If sample dilutions are desired,they may be arranged either vertically orhorizontally, and the degree of hybridiza-tion can then be assessed using imageanalysis software.

The two main drawbacks of the dot-blotanalysis, which yields purely quantitativedata, are: (1) that it lacks the qualita-tive component that accompanies elec-trophoresis; and (2) that the immobi-lization of the samples on a membraneseverely limits the assay’s quantitativecharacter. In order to be truly reliable,a dot-blot analysis must include excel-lent positive and negative controls todemonstrate hybridization specificity, andto gauge any nonspecific binding of theprobe to the filter membrane. Moreover,good internal controls are always in order:equally intense signals should be observedfrom wells into which equal amounts ofpositive control target have been applied.

When attempting this type of blot analysisfor the first time, or when using a newsystem, it is strongly suggested that dilu-tions of the positive control target materialare made in order to determine the linearrange of the assay. For example, it wouldbe useless – quantitatively speaking – ifthe hybridization signals were too intenseto be accurately measured on X-ray film,which also has a defined linear range[17].

6.6High-Throughput Transcription Analysis

The ability to rapidly screen a large numberof samples and simultaneously assay theexpression of as many genes as possible(global analysis of gene expression) hasbecome a reality with the developmentof microarrays. The observed pattern of alarge number of genes that are modulatedunder a defined set of conditions, whichsometimes is referred to as expression


profiling, is perhaps the most commonmicroarray application.

A microarray is typically a glass slide,silicon wafer, or even a plastic substrateupon which very large numbers (currentlyhundreds of thousands) of portions ofindividual gene sequences (genomic orcDNA) have been permanently applied [18,19]. These devices are sometimes referredto as gene chips. It is worth noting thatother microarray-based technologies arecurrently available, such as protein mi-croarrays [19, 20] (commonly known asprotein biochips) and antibody (Ab) mi-croarrays. Consequently, microarray de-signs fall into three categories: (1) genomicarrays, which are used to study the struc-ture and organization of genomic DNA;(2) transcriptome arrays, which are usedto measure gene expression at the level ofRNA synthesis; and (3) proteomic arrays,which are used to measure protein expres-sion and also to study protein interactions.

Originally, each microarray was printedwith sequences representing a uniquetissue. Today, however, multiple-tissuemicroarrays are becoming increasinglypopular, thereby facilitating the simul-taneous assay of several tissues. Thisapproach is analogous to the very pop-ular multiple-tissue Northern blots thatare available commercially from manybiotech suppliers, where RNA from severaltissues has been blotted and is readyfor nucleic acid hybridization. In a way,multiple-tissue microarrays represent ahigh-tech, high-throughput extension ofin situ hybridization, in which geneexpression is assigned to specific cell typeswithin the architecture of a tissue sam-ple. Microarrays are also available withvarious themes, such as a cancer array(sometimes referred to as a cancer panel).These specialized microarrays, as well asmicroarrays printed with broad-ranging

sequences, are designed to provide inves-tigators with as much latitude as possiblein designing their experiments and inter-preting the very large amount of resultantdata.

Microarrays are probed in a very smallvolume (200 μl) of hybridization bufferovernight with labeled cDNA or cRNAfrom two different sources, such as atreated sample and a control sample. EachcDNA probe is labeled with a differentfluorescent dye, most often Cy3 (greenfluorescence) and Cy5 (red fluorescence).These probes are mixed together and usedto cohybridize to targets printed on the mi-croarray (Fig. 7). If Cy3-labeled cDNA andCy5-labeled cDNA hybridize to the samespot on a microarray, the laser-inducedfluorescence of both Cy3 and Cy5 makesthe spot appear yellow. If either probealone hybridizes to a sequence on the ar-ray, then green or red fluorescence willbe observed, which means that the cor-responding gene is expressed in one orthe other sample alone. Thus, it is thefluorescence ratio-based analysis of eachspot on the microarray that provides in-formation concerning the abundance ofparticular transcripts. Although microar-rays are considered high-throughput tools,they are not especially quantitative. Can-didate genes are generally identified byhigh-throughput microarray analysis, afterwhich the behavior of the genes may bemore fully characterized, at least at thetranscriptional level, by very quantitativereal-time PCR.

Although microarray analysis is per-haps the best known format for high-throughput analysis, other methods areavailable in the repertoire of the molecularbiologist. Among these are included theserial analysis of gene expression (SAGE)[21], mRNA differential display [22], am-plified fragment length polymorphisms


RNA Sample 1

cDNA Sample 1

Cy5-labeled cDNA

Isolate RNAAssess purity and

integrity

cDNA SynthesisStabilizes each sample

against degradation

cDNA LabelingAttachment of

fluorescent dyes

DifferentialHybridization

Hybridize to microarray

Image AnalysisSoftware

interpretation of results

HybridizationA sequence on an arraymay hybridize one, both,or neither of the probes.

Cy5 probe = redCy3 probe = green

Both probes = yellowNeither probe = no color

RNA Sample 2

cDNA Sample 2

Cy3-labeled cDNA

Fig. 7 Major steps in microarray analysis.cDNAs are synthesized, labeled, and hy-bridized to an array. Fluorescence detectioncoupled with image analysis provides a greatdeal of information of about patterns of geneexpression in the samples under investigation.

Reproduced with permission from Farrell Jr,R.E. (2010) RNA Methodologies: A LaboratoryGuide for Isolation and Characterization, 4thedn, Elsevier, Academic Press, San Diego, CA,p. 503) [1].

(AFLPs) [23], massively parallel signaturesequencing (MPSS) [24] and, most re-cently, whole-transcriptome deep sequenc-ing (RNA-Seq) [25]. Each of these meth-ods profiles the expression of most (orall) genes simultaneously, without priorknowledge of the identity of those genes.Consequently, any gene can be assayedwithout knowing ahead of time which

genes should be examined, which is in di-rect contrast to all forms of classical PCR,in which gene-specific primers are used totarget only one cDNA species for amplifi-cation. Inasmuch as these newer, highlysensitive methods are describes in detailelsewhere in this volume, the methodsare briefly mentioned here as a point ofreference for additional information.


Total RNA

SMART™cDNA synthesis

ds tester cDNAds driver cDNARsa I digestion

Driver cDNA (in excess)

First hybridization

Tester cDNA with Adaptor 1

a

a, b, c, d + e

bc

a

a, d

b′3′

3′ 5′5′ c

e

b

b

c

d

e

d

Second hybridization: mix samples,add fresh denatured driver, and anneal

Fill in the ends

Add primers Amplify by PCR

no amplification

no amplification

linear amplification

exponential amplification

and

Poly A+ RNAConventionalcDNA synthesis

Teater cDNA with Adaptor 2R

Fig. 8 Suppression subtractive hybridization.In this procedure, the tester and driver cDNAstrands are able to interact with each other invarious combinations, leading to enrichment

of upregulated gene sequences found in thetester cDNA population. Illustration courtesyof Clontech Laboratories (PCR-Select cDNASubtraction System).

6.7Suppression Subtractive Hybridization(SSH)

The global identification of differen-tially expressed genes is also possiblein a non-microarray format, and withoutthe bioinformatics challenges associatedwith RNA-Seq and similar next-generationsequencing-based methodologies. In par-ticular, suppression subtractive hybridiza-tion (SSH) is a method of nucleic acidsubtraction of sequences common to

control and experimental cDNA popula-tions, coupled with the PCR amplificationof uniquely expressed sequences [26, 27].Moreover, the mechanics of the assay(Fig. 8) favor a normalization of differentialexpressed sequences, which means thatthe assay produces an enriched pool ofdifferentially expressed sequences that areall present at a similar concentration, re-gardless of their respective abundancelevels in the original biological material.This situation is highly desirable during


the identification process because, in anon-normalized library, highly abundantcDNAs are much more likely to be clonedcompared to cDNAs of lesser abundance.Thus, the sequencing of 100 clones froma normalized cDNA library will providea much more comprehensive, representa-tive biochemical ‘‘snapshot’’ of the varietyof up- or down-regulated sequences, com-pared to a non-normalized library.

In the present author’s laboratory,the SSH method has been used inseveral applications, and has providedoutstanding results [28, 29], generating amore complete profile of gene expressionthan did the older method of mRNAdifferential display. It might be expectedthat any sequence which differs inabundance by fivefold would be easilysequestered by the subtraction process,but it is not uncommon to detect geneswith as little as a 1.5-fold differencecompared to the control population.

Briefly, SSH involves creating twotester (cDNA from experimental cells)subpopulations ligated to differentadapters, while no adapters are ligatedto the driver (cDNA from the controlcells). Subsequently, two hybridizationsare performed to remove those cDNAscommon to the cDNA tester and driverpopulations, leaving only uniquely ex-pressed cDNAs available for amplificationby PCR; the resulting PCR productsare the differentially expressed genes.The enriched, differentially expressedsequences are now ready for cloning,confirmation of differential status, andsequencing. On completion of thesubtraction-suppression PCR procedures,the subtracted cDNAs are ligated toplasmids, followed by transformationinto Escherichia coli. The individualclones can be plasmid-prepped and, aftersequencing, identified using various

bioinformatics tools (e.g., BLAST™analysis) that are available to the contem-porary molecular biologist. Clearly, SSHrepresents a versatile, low-cost alternativeto microarray technology.

6.8RNAi

A remarkable new tool has emerged thatis able to suppress the expression of spe-cific endogenous genes through the useof double-stranded RNA (dsRNA). Duringthe few short years since the first demon-stration of the power of this techniquein Caenorhabditis elegans [30], and subse-quently in human and other mammaliancells [31, 32], refinements in the method-ology have had the same level of impacton research that PCR did 25 years ear-lier. This new and emerging technologyis known as RNA interference (RNAi); itis also referred to occasionally as ‘‘geneknockdown’’ or ‘‘post-transcriptional genesilencing.’’ It should be noted that RNAiis a patented process, and the commercialuse of this process may require licensingthrough the Carnegie Institute of Wash-ington (www.ciw.edu).

RNAi is a natural phenomenon thatwas considered an oddity when it wasfirst observed in petunias [33, 34]. It isnow known to exist in many organismsas a means of protecting against virusesand other molecular invaders that wouldotherwise plague and wreak havoc witha host genome. This method of protect-ing the integrity of the eukaryotic genomeis highly conserved, and involves the tar-geting of dsRNA entering the cell for itsimmediate destruction. Natural processesmediated by RNAi include the turnoverof wild-type and mutant mRNAs, transla-tional regulation during the developmentof an organism and, undoubtedly, also


other regulatory mechanisms in the cellthat have not yet come to light.

RNAi is an endogenous catalytic path-way that is triggered by dsRNA. The ‘‘trig-ger’’ can occur either naturally, as in thecase of a cellular infection by a dsRNAvirus, or by the intentional introductionof dsRNA to induce a user-directed degra-dation of the complementary transcript(s).The net result of RNAi is the downregula-tion of specific genes by the destruction oftheir mRNA(s). This method of studyingthe effects of gene expression in a cell or inan organism is loosely referred to as reversegenetics, the goal of which is to determinethe consequences for a cell or an organismwhen a protein is not produced. In addi-tion to developmental biology, RNAi hasprofound ramifications in the treatment ofinfectious diseases, and in other diseasesthat result from inappropriate protein ex-pression, such as gain-of-function muta-tions. The major strength of RNAi is thatit permits the study of the function of onegene at a time over an extended period.

Recently, RNAi has emerged as an im-portant mainstream tool for both basic

and applied research, and has already rev-olutionized the area of study known asfunctional genomics. RNAi is ubiquitous ineukaryotes, and currently is a favored toolfor investigating the regulation of geneexpression in plants, animals, and fungi.This technology is becoming increasinglypopular owing to its compatibility withcell culture as well as in vivo research mod-els. Comparatively speaking, the processof RNAi is much faster and far more eco-nomical than creating knock-out animalsin order to study the function of specificgenes. The resulting precision silencing ofspecific genes also makes RNAi an attrac-tive platform for the discovery and devel-opment of life-saving pharmaceuticals.

There are multiple approaches by whichRNAi can be induced, each of whichhas several mechanistic permutations.RNAi is, fundamentally, a two-stepprocess (Fig. 9). The first step involvesone of the master enzymes in the RNAiprocess, a type III endoribonuclease aptlynamed Dicer. This enzyme, a ubiquitousmember of the eukaryotic proteome, isinvolved in the ATP-dependent cleavage

RNAi – Major Steps

dsRNA

siRNA

mRNADegradation

Dicer

RISC

5′ AAAAA

Fig. 9 Major steps in the RNAi process.Double-stranded RNA (dsRNA) from any of anumber of sources, and in any of a numberof configurations, is cut by the enzyme Dicerinto siRNA which, in turn, become part of themulticomponent RISC. This ultimately leads to

destruction of the target mRNA and concomitantdownregulation of the associated gene. Repro-duced with permission from Farrell Jr, R.E. (2005)RNA Methodologies: A Laboratory Guide for Isola-tion and Characterization, 3rd edn, Elsevier, SanDiego, CA, p. 607) [1].


of long dsRNA into 21–23 bp shortinhibitory RNA (siRNA) molecules withcharacteristic 3′ dinucleotide overhangson both strands [35]. Long dsRNA canbe introduced into mammalian cells,where it will become a substrate forDicer, although care must be taken toensure that the dsRNA is not so long asto induce the interferon pathway, leadingto apoptosis. A more prudent choice is touse recombinant Dicer to generate siRNAin vitro, after which these short moleculesare introduced into the cell by transfection.In the second step, siRNA, regardless ofthe source, becomes part of a multicom-ponent nuclease-containing RNA-inducedsilencing complex (RISC). Part of theRISC is an ATP-dependent helicase thatunwinds the double-stranded siRNA, theantisense component of which is now ableto base-pair with the mRNA to be silenced.The formation of a double-stranded regionbetween the antisense component of thesilencing RNA and the cognate mRNAseals its fate. The RISC cuts the mRNAclose to the middle of this transientlyformed double-stranded region [36], afterwhich the mRNA is further degraded,preventing any level of interaction withthe cellular translation machinery.

It is worth noting that there remainsmuch confusion as to the functionalsimilarities and differences betweensiRNA and miRNA. In terms of the netresult, miRNA and siRNA actions areoften indistinguishable; hence, the majordifference is not what these moleculesdo, but rather where they come from.miRNAs result from the endogenoustranscription of genomic DNA; eukaryoticcells are also replete with the nuclearenzyme Drosha, which is responsible forthe initial post-transcriptional processingof naturally occurring nuclear transcriptsthat will mature into miRNA molecules.

In contrast, siRNAs are the result ofeither the processing of long dsRNA,the processing of short hairpin RNA(shRNA) molecules that are producedby expression vector transcription, orby the direct introduction of siRNA viatransfection. Both, siRNAs and miRNAs,mature through the action of one of thecytoplasmic Dicer enzymes, which leadsto their association with a RISC-likecomplex. The major functions of miRNAare translational repression and mRNAcleavage, while siRNAs tend to be asso-ciated with mRNA cleavage alone. Thesubtleties of RNAi are described in muchgreater detail elsewhere in the EMCBMM.

6.9In Vitro Translation

The classical methods used for the analysisof RNA include all of the standard tech-niques described above, and many others.Another aspect of the RNA story, however,is the destiny of those transcripts whichemerge as mature mRNA molecules in thecytoplasm. mRNA biogenesis guaranteesneither translation nor translatability, anda gene is really not ‘‘expressed’’ until afunctional peptide is produced. In order tomore fully characterize gene expression,the extent of translation may be assessedby Western analysis [37], and the resultingdata correlated to the transcriptionalactivity of the corresponding gene(s).

The translation of mRNA into proteinin vitro goes hand-in-hand with the quan-tification of that transcript as a parameterof gene expression. Briefly, mRNA synthe-sized naturally or by in vitro transcriptionis added to a whole-cell lysate contain-ing the components needed to supportthe in vitro translation of those mRNAs.The translation is then performed in thepresence of radiolabeled amino acids or,


more recently, of amino acid labels thatsupport nonisotopic detection. Commonin vitro translation systems include thereticulocyte lysate system and the wheatgerm extract system, and in some cases,the microinjection of message directlyinto a living cell. The pretreatment ofan in vitro translation lysate with mi-crococcal nuclease destroys endogenousmRNA, ensuring de novo protein syn-thesis exclusively from the experimentalmRNA added to the system. The samein vivo requirements for 5′ cap, initi-ation codon, and polyadenylation applyhere if the experimental introduced mes-sage is to be translated efficiently. Theprotein products are then analyzed asusual by using Western analysis, bio-logical assay, immunopurification, or re-lated techniques. This particular aspectof biotechnology is attractive for a num-ber of different applications, including thestudy of naturally occurring animal andplant mRNAs, transfection experiments,the characterization of mRNA products,protein engineering, the screening andanalysis of mutants, and DNA-, RNA-, orprotein-binding studies.

7Summary

The characterization of RNA from bio-logical sources is a central action of pro-found significance in molecular biology.Gene expression is frequently analyzedusing blot analysis, by solution hybridiza-tion methods, or by any of a number ofRT-PCR variations. Increasingly, many ofthe classical RNA analysis methodologieshave become sidelined or even passedover altogether because of the power ofPCR, and the fact that the methods de-scribed here have revolutionized many

aspects of biotechnology is not in doubt.RNA is analyzed to answer transcrip-tion questions, and the tools describedin this chapter are currently in widespreaduse to this end. The method by whichRNA is isolated, the downstream tech-nique(s) used to analyze the RNA, andthe level of sensitivity associated with thattechnique are all variables that must beweighed up before any laboratory investi-gations are undertaken. RNA methodolo-gies have a non-negotiable requirementfor high-quality starting materials if theensuing data are to be representative, toaccurately reflect the cellular biochemistry,and to provide insight into the subtletiesof gene regulation.

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Part I Analytical Methods - Wiley-VCH I Analytical Methods ... Chaotropic lysis buffers disrupt the cell and organelle ... RNA type Symbol Basic function Prokaryotic Eukaryotic