Scribd
Upload
81 views7 pages

RT-PCR: Methods and Principles

RT-PCR is a technique used to detect RNA expression levels that involves first converting RNA to cDNA using reverse transcriptase, then exponentially amplifying the cDNA using PCR. It has become the benchmark method for detecting and comparing RNA levels, displacing Northern blot. RT-PCR can be performed as either a one-step or two-step reaction, with the one-step approach combining cDNA synthesis and PCR amplification in a single tube and two-step separating these steps into different tubes.

Uploaded by

Vladimir Magnus
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
81 views7 pages

RT-PCR: Methods and Principles

RT-PCR is a technique used to detect RNA expression levels that involves first converting RNA to cDNA using reverse transcriptase, then exponentially amplifying the cDNA using PCR. It has become the benchmark method for detecting and comparing RNA levels, displacing Northern blot. RT-PCR can be performed as either a one-step or two-step reaction, with the one-step approach combining cDNA synthesis and PCR amplification in a single tube and two-step separating these steps into different tubes.

Uploaded by

Vladimir Magnus
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

Reverse Transcriptase PCR

(RT PCR)
Background
RT-PCR - one of many variants of
polymerase chain reaction.
This technique commonly used in
molecular biology to detect RNA
expression levels.
RT-PCR often confused with real-time
polymerase chain reaction (qPCR) by
students and scientists; they are
separate and distinct techniques.
Bit of history
the discovery of reverse transcriptase
(enzyme) during the study of viral replication
of genetic material led to the development of
RT-PCR.
RT-PCR displaced Northern blot as the method
of choice for RNA detection and quantification.
RT-PCR has risen to become the benchmark
technology for the detection and/or
comparison of RNA levels.
Principles of RT-PCR
the RNA template is first converted
into a complimentary DNA (cDNA)
using a reverse transcriptase.
The cDNA is then used as a template
for exponential amplification using
PCR.
Principles of RT-PCR
One step Vs Two step RT-
PCR
The quantification of mRNA using RT-
PCR can be achieved as either a one-
step or a two-step reaction.
In the one-step approach, the entire
reaction from cDNA synthesis to PCR
amplification occurs in a single tube.
The two-step reaction requires that the
reverse transcriptase reaction and PCR
amplification performed in separate
tubes.

You might also like