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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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