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OF THIS PRESENTATION
What is RT-PCR?
How does it works?
What does it tell us?
Difference between RT-PCR and
What work is done through RT-PCR?
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
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.
Reverse transcriptase discovery in 1977 led
to the development of RT-PCR
since it displaced the Northern blot method
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
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
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
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
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
The RT-PCR products can then be analyzed with gel electrophoresis.
Two step RT-PCR
Combine template RNA, primer, dNTP mix, and nuclease-
free water in a PCR tube.
Add RNase inhibitor and reverse transcriptase to the PCR
Place PCR tube in thermal cycler for one cycle that includes
annealing, extending and then inactivating reverse
Proceed directly to PCR or store on ice until PCR can be
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:
The RT-PCR products can then be analyzed with gel
electrophores contamination due to more frequent sample
Comparison Two-Step Procedure One-Step Procedure
Prime first-strand cDNA
Choice of primer
•Choice of amplification
•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
premixed with reverse
•Fewer pipetting steps and
reduced chances of
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
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
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
Offer particular help to researchers who may have limited
experience in isolating nucleic acids
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
( Urology Volume 43, Issue 6, June 1994, Pages 765–775)
CCHF virus variants in Pakistan and Afghanistan: Emerging diversity
(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)