RT- PCR: Reverse transcriptase polymerase chain reaction

Ø (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called
complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR).

Ø It is primarily used to measure the amount of a specific RNA.

Ø This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or
quantitative PCR (qPCR).

Ø Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in
research and clinical settings.

The combined RT-PCR and qPCR technique has been described as quantitative RT-PCR or real-time RT-PCR
(sometimes even called quantitative real-time RT-PCR ), has been variously abbreviated as qRT-PCR, RT-qPCR,
RRT-PCR, and rRT-PCR. I
ü Template: RNA

ü Using reverse transcriptase enzyme (RNA → cDNA)

ü One step & two step RT-PCR

ü Reverse transcriptase
- AMV reverse transcriptase (Avian myoblastosis virus)
- MMLV reverse transcriptase (Moloney murine leukemia virus)

ü tissue sample → RNA →cDNA→ PCR

One step versus two steps RT-PCR
One step versus two steps RT-PCR
 Real-Time Polymerase Chain Reaction (qRT-PCR)
In RT-PCR, The RNA population is converted to cDNA by v Common applications of RT-PCR include detection
reverse transcription and then the cDNA is amplified by PCR. of expressed genes, examination of transcript
The cDNA amplification provides opportunities to further variants, and generation of cDNA templates for
study the original RNA species, even when they are limited in cloning and sequencing
amount or expressed in low abundance.
Real-Time Polymerase Chain Reaction (qRT-PCR)
Real-Time Polymerase Chain Reaction (qRT-PCR)
Real-Time Polymerase Chain Reaction (qRT-PCR)
- Simultaneous amplification of target DNA by PCR, and detection
- Target DNA is amplified and labeleled with fluorescently tagged probes
- The accumulating product is monitored and measured by detecting the
fluorescence
- More then one targets can be amplified and detected (multiplex)
- Each target is labeled with different dyes

Fluorescence Setting
Target Fluorophore (dy) Channel
RdRp gene FAM Green
N gene JOE (ABI)/VIC (CFX96) Yellow
E gene Texas Red Orange
IC Cy5 Red
No QUENCHER, Reference dye None