This document provides an overview of reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is a laboratory technique that combines reverse transcription of RNA into cDNA and amplification of specific DNA targets using PCR. It allows for detection and quantification of RNA. The document describes the basic principles and components of RT-PCR, including reverse transcriptase, primers, and DNA polymerase. It also discusses the differences between one-step and two-step RT-PCR methods and outlines several applications of RT-PCR in research and disease diagnosis.

1. Name : Sankalpa Sahoo
Regd No : 200705180030
Session : 2020 – 2022
Guided By – Dr Gagan Kumar Panigrahi
School of Applied Sciences
(SoAS)
BHUBANESWAR
RT - PCR

2. CONTENTS
 Introduction of RT-PCR
 PCR vs RT-PCR
 Basic principles
 Components used in RT-PCR
 Types of RT-PCR
 Applications
 References

3. INTRODUCTION OF RT-pCR
 RT-PCR stands for –
Reverse Transcription Polymerase Chain Reaction
 It is one of many variants of the conventional PCR
 The discovery of reverse transciptase during the study of viral
replicaton of genetic material led to the development of RT-PCR
 This technique is commonly used to detect RNA expression
 In this technique complimentary DNA (cDNA) transcripts are
created from a given RNA to qualitatively detect gene expressions
 It is a combinaton of reverse trancription of RNA into DNA and
amplification of specific Dna tragets using polymerase chain reaction

5. PCR vs RT-PCR
 Traditional PCR is used to exponentially amplify target DNA
sequences
 RT-PCR is used to reverse transcribe mRNA to cDNA and then
amplify it using traditional PCR
 In PCR, a double-stranded DNA molecule is used as the starting
template
 In RT-PCR, a single-stranded RNA molecule is used as the
starting template
 While PCR is less sensitive, RT-PCR is more sesitive
 Since mRNA is involved in translation, RT-PCR can give a
measurement of gene expression that PCR can’t

7. Basic principles of rt-pcr
 RT-PCR involves total RNA isolation, reverse transcription (RT)
and PCR.
 RNA is easily degraded since RNase is very hard to inactivate.
So several precautions are taken.
 Total RNA isolation can be done by TRI reagent, Chloroform,
isopropanol, 75% ethanol and molecular grade and RNase-free
water.
 Reverse transcription is done by taking total RNA with reverse
transcriptase, dNTP mixture, RNase inhibitor, Oligo dT primer and
RNase-free water.
 PCR involves template cDNA from the RT, upstream and
downstream primers, heat-stable DNA polymerase like Taq
polymerase, dNTP mixture, agarose, ethidium bromide solution

8. components used in RT-pcr
 TRI reagent – It is a mixture of guanidine thiocyanate and
phenol in a monophase solution. It dissolves DNA, RNA and
protein.
 Reverse transcriptase (RTase) – They are used by certain
viruses to replicate their genomes. We use M-MLV RTase and
AMV RTase in RT-PCR.
 M-MLV – Moloney-murine leukemia virus
 AMV – Avian myeloblastosis virus
 dNTP – deoxynucleotide triphosphate. They are four types i.e.
dATP, dCTP, dGTP, dTTP. Using dNTP provides single bases
ready to go into DNA and double it.

9.  RNase inhibitor (RI) – It is a leucine-rich repeat protein which
forms extremely tight complexes with RNase. Murine is used for
cDNA synthesis.
 Oligo dT primer – It is a single-stranded sequence of
deoxythymine (dT) used for priming reactions catalysed by RTases.
The transcript is primed in the poly(A) tail of mRNA.
 Taq polymerase – It is a heat-stable DNA polymerase I named
after Thermus aquaticus.
 Agarose – It is a polysaccharide, generally extracted from some
red seaweed. It is used to separate DNA by electrophoresis.
Agarose gel have relatively large pore size.
 Ethidium bromide – It is commonly used as a fluorescent tag
(nucleic acid stain) in agarose gel electrophoresis. When exposed
to UV light, it will fluoresce with an orange colour.

10.  This method is more sensitive than the one-step method.
 Primers in this case don’t need to be sequence specific.

11. One-step RT-PCR
 All reaction components are mixed in one tube and the
reaction is carried out.
 It offers simplicity and convenience. It also minimizes the
chances of contamination.
 The primers must be sequence specific.
 The resulting cDNA can’t be used to detect multiple
informations from a single RNA sample.

12. applications
 RT-PCR is a relatively simple, inexpensive, extremely sensitive
and specific way to determine the expression level of target
genes.
 It is widely used in biomedical science research to detect
genetic diseases and semi-quantitatively to determine the
abundance of different RNA molecules within a cell.
 In this method a very low copy number of RNA molecules can
be detected.
 It is also used in research methods like expression of Gal
genes in yeast cells.

13.  Gene insertion – It is also very useful in insertion of eukaryotic
genes into prokaryotes.
 It is used to study genetic materials of retroviruses like HIV and
other RNA containing viruses like Influenzavirus A.
 Cancer detection – This use of RT-PCR is under serious study
and research. Scientists are studying how to effectively use this
method in detecting cancers.
 Genetic disease diagnosis – It can be used to detect diseases
like Bird flu- H7N9, Lesch-Nyhan syndrome, COVID-19, Ebola etc.

14. REFERENCES
 Reverse transcription polymerase chain reaction
(https://en.wikipedia.org/wiki/Reverse_transcription_polymeras
e_chain_reaction)
 Introduction of RT-PCR
(http://www.slideshare.net/Shababmehebub/introduction-of-rt-
pcr-69566089)
 RT-PCR : One-step vs Two-step
(https://www.thermofisher.com/in/en/home/brands/invitrogen/m
olecular-biology-technologies/spotlight-articles/onestep-vs-
twostep-rtpcr.html)
 Application of RT-PCR and real-time PCR
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087796)