Vermont Genetics NetworkVermont Genetics NetworkMicroarray Outreach ProgramMicroarray Outreach Program

Large Scale Gene Large Scale Gene Expression with Expression with

DNA MicroarraysDNA Microarrays

Vermont Genetics Network (VGN)

Founded at the University of Vermont in 2001 through an NIH BRIN grant and renewed in 2005 through an NIH INBRE grant

Purpose:

• Encourage biomedical research in Vermont• Create a “network” of researchers and students • Give outreach lectures to 4-year institutions • Provide research grants to faculty and students • Mentoring for students interested in research

VGN Microarray Outreach Program •Develop microarray outreach module.

•Introduce microarray technology to VT colleges.

•Team of scientists to serve as instructors

Tim Hunter, Pat Reed, Janet Murray, Scott Tighe

Why Gene Expression using Microarray?Why Gene Expression using Microarray?

• Up and coming modern technology- complicated

• Not affordable to all colleges

• Well adapted mini coarse with a “big picture”

• Can use many model organisms

• Employs large scale bioinfomatics-one of VGN’s goals

Desired Outcomes of Microarray moduleDesired Outcomes of Microarray module

• Learn new techniques that are state of the art• Improve laboratory skills• Build self confidence• Creates new opportunities:

• Internships• Job offers due to experience in new technology• Build contact networks

MicroarraysMicroarrays

• Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of features (usually between 500 up to 7.5 million)

• Using a conventional hybridization process, the level of expression of genes is measured

• Microarrays are read using laser-based fluorescence scanners

What are Microarrays?

What are Microarrays?

InstrumentationAffymetrix GeneChip System3000-7G Scanner450 Fluidic Station640 Hybridization Oven

Affymetrix GeneChip

$400 each

$300,000

Arabidopsis ATH1 Genome Array

This GeneChip contains 500,000 DNA oligos comprising 24,000 genes-

The image on the left is a full scan of the GeneChip while the image on the right is a 1000X zoom of a small area.

Why use Microarrays?Why use Microarrays?

• What genes are Present/Absent in a cell?

• What genes are Present/Absent in the experiment vs. control?

• Which genes have increased/decreased expression in experiment vs. control?

• Which genes have biological significance?

Microarray Applications

• Identify new genes implicated in disease progression and treatment response (90% of our genes have yet to be ascribed a function)

• Assess side-effects or drug reaction profiles

• Extract prognostic information, e.g. classify tumors based on hundreds of parameters rather than 2 or 3.

• Identify new drug targets and accelerate drug discovery and testing

• Gene Discovery-– Assigning function to sequence– Discovery of disease genes and drug targets– Target validation

• Genotyping

– Patient stratification (pharmacogenomics)– Adverse drug effects (ADE)

• Microbial ID

Microarray Applications

Why analyze so many genes?Why analyze so many genes?

• Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function.

• Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

The 2008 Marlboro ExperimentsThe 2008 Marlboro Experiments

• What is your experiment

• What is your question or hypothesis

• What is your organism

• Will you be able to keep the study system stable and comparable

• How many duplicates/samples will be enough

Experimental DesignExperimental Design Is this a “fishing expedition” or a hypothesis-based experiment?

Choice of reference (control): Common reference, Non-treated, WildtypeAs important as the experimental samples

Number of replicates (required!!!): How many are needed ?How many are affordable?Pooling of samples???

Normal vs. NormalNormal vs. Normal Normal vs. TreatedNormal vs. Treated

UP

Down

Scatter plots reveal changes between samples

Laboratory Procedures and ConsiderationsLaboratory Procedures and Considerations

DNA (genes)

messenger RNA

Protein (effector molecules)

The Central DogmaThe Central Dogma

[Expressed Genes = mRNA][Expressed Genes = mRNA]

Consider the characteristics of your model organism!Consider the characteristics of your model organism!

E. coli

~ 1 x 3 m

Yeast

~ 5 m dia.Human

~ 1.7 m

1 chromosome

4 x 10 6 bp

16 chromosomes

12 x 10 6 bp

23 chromosomes

3.3 x 10 9 bp

~ 4,377 genes ~ 30, 000 genes~ 5,726 genes

Arabidopsis Characteristics….Arabidopsis Characteristics….• How many chromosomes?

• What is expected RNA yields?

• What are the problems that investigators encounter routinely?

• What genes do you expect will change?

• Check the databases: http://www.arabidopsis.org/ Netaffx-Affymetrix

First step –Get good First step –Get good totaltotal RNA RNA

• Five types of RNA

– Ribosomal- [rRNA-5s, 5.8s 16s, 18s, 25s 28s]

– Transfer- [tRNA]-protein synthesis]

– Messager- [mRNA-Poly adenylated]

– Micro- [miRNA-RNA control]

– Small Nuclear- [snRNA]

We need total RNA –We need the rRNA as an indicator of quality

Creating TargetsCreating Targets

Reverse Transcriptase

in vitro transcription

mRNA

cDNA

cRNA

cDNA2nd Strand

1st Strand

GeneChip

The steps of a The steps of a microarray microarray experimentexperiment

Quality Control Is Quality Control Is VeryVery Important because Important because microarray is very expensive!microarray is very expensive!

• RNA purity and integrity Nanodrop spec Bioanalyzer

• cDNA synthesis efficiency

• Efficient cRNA synthesis, labeling, and fragmentation

• Target evaluation with Test Chips

RNA-DNA Hybridization, Staining, and ScanningRNA-DNA Hybridization, Staining, and Scanning

Labeled sample

Inject and Hyb 16hr

Stain and scanExamine and Analyze…..

Data analysis is difficult and time consuming and can takes weeks and months due to the amount of data

Data Analysis:

General Techniques and Guidelines for Extracting Total RNA

Why Total RNA?Why Total RNA?• Not all transcripts have poly A tail

• RNA assessment is more determinative due to rRNA subunit peaks when running a gel or Bioanalyzer

• Recovery of special RNA’s such as MiRNA

• mRNA recovery kits also recover rRNA anyway

General RNA HandlingGeneral RNA Handling

Reagents and Equipment-Considerations:

All reagents MUST be RNase-free

Use gloves that are periodically treated with RNase Zap

Perform as must work in a hoodBiosafetyLaminar flow PCR hoodDO NOT use a fume hood

Use aerosol resistent pipet tips

Prepare all surfaces and pipets by treating with RNAse Zap

All utensils [scissors, scalpels, tweezers] should be scrubbed clean, sprayed with RNase Zap, soaked in ETOH and flame sterilized before a surgery

Prepare daily aliquots of RNase-free water. I aliquot 10-20 tubes each week and discard half way through the day.

Diethylpyrocarbonate [DEPC]-treated water is NOT an inhibitor for RNases, but rather DEPC is a chemical added to water to eliminate RNases. After autoclaving [or when purchased], no DEPC resides in the water.

When opening and closing tubes, be careful not to bump the inner rim of your tubes.

General RNA HandlingGeneral RNA Handling

RNA Extraction SystemsRNA Extraction Systems

RNA Isolation and Purification Systems-[Column-based]RNA Isolation and Purification Systems-[Column-based]RNeasy Micro kit [74004]

Small elution volumes 10-15ul for 10ugGood for FACS samples, LCM, or limited cellOn-column DNase treatment

RNeasy Mini Kit [74104]Standard Elution volume of 30-50ul for 100ugGeneral use columnOn column DNase treatment Lipid or Fiber kits too

RNeasy Midi and Maxi KitLarge elution volumes 150-800ul for 1mg of RNA

USB Corp Prep-Easy Kit [78766]A knock off of the RNeasy Mini kitElution volume of 30ulCheaper than RNeasy and comes with DNase

Zymo Corp

Not recommended-poor recoveries in our hands

• RNeasy and USB system are a 4M guanidine isothiocyanate [GCN] chaotopic salt system that strongly denatures RNases-no phenol

• RNA is precipitated with ethanol and bound to silica [Si-O-H], washed, and eluted with water.

PROS CONSEasy to perform Will not isolate MiRNA

No precipitation rxn Will not isolate RNA <200bpVery clean RNA Lower yield than TrizolCan extract in RLT Buffer Lipids can interfere and inhibitDNase on the column Poor storage integrity at -80Compatible with FastPrep Abrasives can end up in final sampleCompatible with Shedder column Heavy DNA contamination –bad[neutrophils, PMN] Salts may be left behind

RNA Isolation and Purification Systems-[Column-based]RNA Isolation and Purification Systems-[Column-based]

Silica Columns-how they workSilica Columns-how they work

Once water is introduced, the electrostatic bond is “broken” and RNA is immediately released back into the “polar” water

Silica Column-based Applications [RNeasy]…continuedMultiple applications of the same elution water increases yield

Be sure not to “spin-out” your DNase before adding RW1

Do not spin with column with cap “open” as described in directions

The final dry spin can be done for 3 minutes

Some columns allow a final rinse with 80% ETOH to reduce salt carryover- Ask your tech support

260/280 ratios are often above 2.00

Can add 0.5ul of Superase™ to collection tube if imparitive-Under special circumstances

If using MinElute or MicroElute columns-be aware of the o-ring-it catches and retains liquid that can get into your final sample

Extracting RNAExtracting RNA

Extracting RNA from Tissue- Extracting RNA from Tissue- Types of homogenizationTypes of homogenization• Traditional mortor and pestle with LN2

• Liquid Nitrogen is not always RNase-free• Difficult to make sterile and RNase-free

• Poly-tron with new tips or RNase-free blades• Not optimized for very small quantities• Sometimes difficult to make RNase-free

• Mini motorized pestle• With or without abrasive• All RNase-free disposable

• Impactor [bio-pulverizer]• French Press• Biomasher columns

• FastPrep System and Mini-bead beater • Automated, fast, RNase-free• Uses screw cap 2ml tubes, 15ml and 50ml and • optional abrasive for homogenizing tough tissue• Excellent for bacterial extractions

Extracting RNA from Tissue- Extracting RNA from Tissue- Types of homogenizationTypes of homogenization

Beware:

Some beads are silica-based and can bind nucleic acids in the presence of binding buffers

Quality Control of RNAQuality Control of RNA

• Consider running an RNase-free gel– Look for gDNA – Look for rRNA bands

• We use the E-gel routinely– Fast, RNase-free agarose gel

Quality Control of RNAQuality Control of RNA

Quality Control of RNAQuality Control of RNA

• Measuring your RNA on the Nanodrop– Look carefully at the trace! – Can not distinguish DNA from RNA– Can not distinguish degraded RNA from “good” RNA– Quantitative interferences can lead to questionable downstream

results

Good RNA

RNA prep with mostly gDNA from Neutrophils

Good vs Degraded RNAGood vs Degraded RNA

Thing are not always as they appear- These look great on the nanodrop… but…. are they?

Quality Control of RNAQuality Control of RNA

Quality Control of RNAQuality Control of RNA ….expected yield? ….expected yield?• Expected yield is very important!!!

• Just getting “enough” RNA is not always ok

• Actively growing mammalian cells contain 1-10 pg/cell

• Calculate your expected RNA recovery

• If you are way below your expected yield than….– Selectively recovered RNA from weak or apoptotic cells– Selectively recovered RNA from G0 or G2M

***Will significantly impact gene expression data***

We see apx 500 ng of RNA per 1mg of tissue

Problematic Nanodrop TracesProblematic Nanodrop TracesSample is NOT 183 ng/ul most likely much less

272 nm peak is skewing data

260 272

How will this affect downstream processes such as RT-qPCR if one assumes equal RNA input to a cDNA reaction?

Marlboro 2007….the fun has just begun

How do you use this thing?....

Oh, no…where’s my RNA? Oh, no…where’s my RNA?

Sites that have participated:Sites that have participated:

Marlboro College Marlboro College St. Michael’s CollegeSt. Michael’s College

Johnson State College Johnson State College Middlebury College Middlebury College

Green Mountain College Green Mountain College Norwich University Norwich University

Castleton State College Castleton State College Lyndon State College Lyndon State College University of Vermont University of Vermont