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RT-PCRPRESENTED BY:
IQRA WAZIR
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Conventional and qRT-PCR
PCR vs RT-PCR
Types of RT-PCR & Primer Designs
Protocol & Application of RT-PCR
Conventional Reverse Transcription-PCR (RT-PCR)
Variant of polymerase chain reaction (PCR)
Commonly used in molecular biology to detect RNA expression
RT-PCR is often confused with real-time polymerase chain reaction (qPCR)
However, they are separate and distinct techniques.
RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA)
transcripts from RNA.
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Reverse Transcription Real-Time-PCR or Quantitative Reverse Transcription-PCR (RT-qPCR)
Quantitative reverse transcription PCR (RT-
qPCR) is used when the starting material is
RNA.
RNA is first transcribed into
complementary DNA (cDNA) by reverse transcriptase from
total RNA or messenger RNA
(mRNA).
The cDNA is then used as the template for the
qPCR reaction.
RT-qPCR is used in a variety of applications
including
Gene expression analysis,
RNAi validation,
microarray validation,
pathogen detection,
genetic testing, and
disease research
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Reverse Transcription Real-Time-PCR or Quantitative Reverse Transcription-PCR (RT-qPCR)
Basics of RT-qPCR:
Isolate RNA from samples
Reverse Transcription
Pick Reference
GeneDesign Primers Run qRT-PCR
Fluorescent signal (eg. Taqman,
SYBERGreen)
Acquire signal at end of each
cycle
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Difference between RT-PCR and Traditional PCR
Traditional PCR is used to
exponentially amplify target DNA
sequences.
RT-PCR is used to reverse transcribe
mRNA to cDNA and then amplify this result using traditional PCR.
Since mRNA is the message that
is sent for translation, RT-
PCR can give us a measurement of gene expression
that PCR cannot.
The cDNA produced does not contain the
introns that DNA does; therefore RT-PCR can provide us with genes that may
be inserted into prokaryotes (which cannot and do not splice the introns
out).
This technique is also used for diagnosing
genetic diseases as well as
studying certain viruses whose
genetic information are
exclusively composed of RNA.
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Types of RT-PCROn the basis of the steps involved there are two types of RT-
PCR.
One- Step RT-PCR Two-Step RT-PCR
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One-Step RT-PCR• All reaction components are mixed in one tube prior to initiation of the
reaction.• Although one-step RT-PCR offers simplicity and convenience and
minimizes the possibility for contamination, the resulting cDNA cannot be used for detecting multiple messages from a single RNA sample as in two-step RT-PCR.
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Two-Step RT-PCR• Traditionally, RT-PCR involves two steps: the RT reaction and PCR
amplification which can be simple PCR or qPCR. • RNA is first reverse transcribed into complementary DNA ( cDNA ) using
an enzyme, reverse transcriptase. • The resulting cDNA is used as templates for subsequent PCR
amplification using primers specific for one or more genes.
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Comparison of One-Step & Two-Step RT-PCR
Two-Step Procedure
One-Step Procedure
Prime first-strand cDNA with:
Oligo(dT) primer Random hexamers Gene-specific primers
Gene-specific primers
Provides Flexibility Choice of primer Choice of amplification
system Ability to save some RNA
sample for later use Ability to optimize for
difficult RT-PCR (combine with Platinum® enzymes for higher specificity or combine with Platinum® Pfx for greater fidelity)
Convenience Amplifcation enzymes
premixed with reverse transcriptase
Fewer pipetting steps and reduced chances of contamination
High sensitivity
Recommended uses: Ideal for detection or quantifying several messages from, a single sample
Ideal for analysis of large numbers of samplesIdeal for real-time quantitative
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Primers Categories:
Primers fall into three categories:
Oligo-dT primers: will specifically anneal to
polyA tails found on most eukaryotic mRNAs. This
type of primers cannot be used with prokaryotic
RNA.
Random hexamers primers: have the
ability to anneal to all types of RNA without
knowledge of sequence.
Gene Specific primers: cannot be used if we want to
prime cDNA synthesis from all the RNAs in the
cell.
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Protocol• RT-PCR can be carried out by the:– One-step RT-PCR protocol – Two-step RT-PCR protocol
• Note: Use only intact, high quality RNA for the best results. Be sure to use a sequence-specific primer.
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One-step RT-PCROne-step RT-PCR
take mRNA targets (up to 6 kb) and subjects them to
reverse transcription and then PCR
amplification in a single test tube.
Select a one-step RT-PCR kit, which
should include a mix with reverse
transcriptase and the PCR system such as Taq DNA Polymerase and a proofreading
polymerase.
Obtain all necessary materials, equipment and instruments (kits
should include a detailed list of
necessary items).
Prepare a reaction mix, which will include dNTPs,
primers, template RNA, necessary
enzymes and a buffer solution.
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One-step RT-PCRAdd the mix to a
PCR tube for each reaction. Then add the
template RNA.
Place PCR tubes in the thermal cycler to begin
cycling. The first cycle is
reverse transcription to
synthesize cDNA.
The second cycle is initial denaturation. During this
cycle reverse transcriptase is
inactivated.
The next 40 to 50 cycles are
the amplification
program, which consists of three
steps: (1) denaturation, (2) annealing, (3) elongation.
The RT-PCR products can
then be analyzed with
gel electrophoresis.
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Two-Step RT-PCRTwo-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR.
This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR.
Use only intact, high quality RNA for the best results. The primer for two-step does not have to be sequence specific.
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Two-Step RT-PCRStep one• Combine template RNA, primer, dNTP mix, and nuclease-free water
in a PCR tube.• Add RNase inhibitor and reverse transcriptase to the PCR tube.• Place PCR tube in thermal cycler for one cycle that includes
annealing, extending and then inactivating reverse transcriptase.• Proceed directly to PCR or store on ice until PCR can be performed.
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Two-Step RT-PCR
Step two
Add a master mix (containing
buffer, dNTP mix, MgCl2,
Taq polymerase and nuclease-free water) to
each PCR tube. (If q-PCR is to be performed
for amplification
use q-PCR master mix).
Add appropriate
primer.
Place PCR tubes in
thermal cycler for 30 cycles of
the amplification
program, which includes three
steps: (1) denaturation, (2) annealing,
(3) elongation.
The RT-PCR products can
then be analyzed with
gel electrophoresis.
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Crucial Aspect & Controls of RT-PCR experiment
• DNA contamination must be avoided• To avoid DNA contamination, two approaches can be used:1. Treat the sample with DNase (A deoxyribonuclease is any enzyme that
catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA).
2. Use in the reaction a negative control which does not contain reverse transcriptase enzyme (-RT).
• If the DNA is effectively eliminated from the starting material (RNA sample):
1. Amplification using “-RT” cDNA template should show no product 2. “+RT” cDNA template should result in a product.
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Applications of RT-PCRResearch methods• For example, Lin et al. used qRT-PCR to measure expression of Gal
genes in yeast cells.
Genetic Disease Diagnosis• RT-PCR can be used to diagnose genetic disease such as Lesch–
Nyhan syndrome (juvenile gout, is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), produced by mutations in the HPRT gene located on the X chromosome).
• Also can be used as a test for bird flu- H7N9.
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Applications of RT-PCR• Gene Insertion• RT-PCR can also be very useful in the insertion of eukaryotic
genes into prokaryotes. • RT-PCR is commonly used in studying the genomes of viruses
whose genomes are composed of RNA, such as:1. Influenza virus and 2. Retroviruses like HIV.
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Applications of RT-PCRCancer Detection• Scientists are working on ways to use RT-PCR in cancer
detection to help improve prognosis, and monitor response to therapy
RNA extracts• RT-PCR is used to test RNA extracts from different tissues
also for the presence of a particular transcript in order to determine the expression pattern of a gene.
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Applications of RT-PCR• Relative and absolute quantification of gene expression.• Validation of DNA microarray results.• Variation analysis including SNP discovery and validation.• Counting bacterial, viral, or fungal loads, etc.
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YOU CAN ASK QUESTIONS!
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