MS-I (ZOOLOGY)

OBJECTIVESOF THIS PRESENTATION

What is RT-PCR? How does it works? What does it tell us? Applications Difference between RT-PCR and

Conventional PCR What work is done through RT-PCR?

INTRODUCTION RT-PCR stands for Reverse Transcription-Polymerase

Chain Reaction. It is a technique used in genetic studies that allows the detection and quantification of mRNA.

Sensitive method that shows whether or not a specific gene is being expressed in a given sample.

RT-PCR is often confused with (qPCR) by students and scientists alike. 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, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes.

DIAGRAMMATIC REPRESENTATION

HISTORY Reverse transcriptase discovery in

1977 led to the development of RT-PCR since it displaced the Northern blot

 method

PRINCIPLE In RT-PCR, the RNA template is first converted into a 

complementary DNA (cDNA) using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR.

In the one-step approach, the entire reaction occurs in a single tube.

In two-step reaction the reverse transcriptase reaction and PCR amplification be performed in separate tubes.

  One-step RT-PCR vs. two-step RT-PCR

https://en.wikipedia.org/wiki/File:One-step_vs_two-step_RT-PCR.jpg

PROTOCOL One step RT-PCR 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.

Prepare a reaction mix, which will include dNTPs, primers, template RNA, necessary enzymes and a buffer solution.

Add 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:

Denaturation annealing elongation The RT-PCR products can then be analyzed with gel electrophoresis.

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PROTOCOL Two step RT-PCR Step 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.

Step two Add a master mix (containing buffer, dNTP mix, MgCl2, Taq

polymerase and nuclease-free water) to each PCR tube. Add appropriate primer. Place PCR tubes in thermal cycler for 30 cycles of the amplification

program, which includes three steps: denaturation annealing elongation The RT-PCR products can then be analyzed with gel electrophores

contamination due to more frequent sample handling

Comparison Two-Step Procedure One-Step ProcedurePrime 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 samples•Ideal for real-time quantitative RT-PCR

TECHNICAL ISSUE While performing RT-PCR One common

difficulty is contamination of the sample with unwanted genetic material that could also be replicated, producing a significant amount of the wrong DNA.

That scenario makes sample preparation critical for both PCR and

RT-PCR

Solution So manufacturers have developed several products and kits to

keep this step unsullied. Ambion, Amersham Pharmacia Biotech, Applied Biosystems,

BIO 101, CLONTECH, Roche Molecular Biochemicals, and Sigma-Aldrich, among others, also produce kits for simplifying extraction and cleanup of DNA and RNA prior to PCR.

Offer particular help to researchers who may have limited experience in isolating nucleic acids

LITERATURE WORK Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between

Infectious and Noninfectious Enteric Viruses in Water Samples (Appl. Environ. Microbiol. July 2010 vol. 76 no. 13 4318-4326)

Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay

( Urology Volume 43, Issue 6, June 1994, Pages 765–775)

 CCHF virus variants in Pakistan and Afghanistan: Emerging diversity and epidemiology

(Journal of Clinical Virology Volume 67, June 2015, Pages 25–30)

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

(J. Clin. Microbiol. March 1992vol. 30 no. 3 545-551)

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