Introduction: o 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. RT_PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, real time PCR is used to quantitatively measure the amplification of DNA using fluorescent probes. o RT-PCR is a variation of standard PCR that involves the amplification of specific mRNA obtained from small samples. In RT-PCR, reverse transcriptase and an RNA sample are used in addition to the standard PCR reagents. RT-PCR is a common virology diagnostic method and is frequently combined with quantitative real-time PCR (q-PCR), which is widely used to quantify RNA transcript levels in cells and tissues.

1. RT_PCRMISHAL TAHIR
DNA RECOMBINANT
TECHNOLOGY
MS/Zoo-1

2. Definition
Reverse transcription of RNA into DNA
called complementary DNA or cDNA
It uses an enzyme called reverse transcriptase
to change a specific piece of RNA into a
matching piece of DNA and amplification of
specific DNA targets

3. Principle of RT-PCR
 RNA is first reverse-transcribed into cDNA
 cDNA then used as the template for PCR amplification
 Primer used for cDNA synthesis can be either:
Non–sequence-specific primers
o Random hexamers
o Oligo-dT primers
Sequence-specific primers
o gene specific primers

5. One-step vs. two-step RT-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 is performed in a separate tube.
https://upload;imedia.org//commons/8/80/One-step_vs_two-step_RT-PCR.jpg
Figure 1.1: One-step vs. two-step RT-PCR

6. https://www.researchgate.net/profile/One-step-and-two-step-methods-of-RT-PCR.png
Figure 1.2: One-step and two-step RT-PCR

8. Real-time PCR (qPCR )
 A technique used to
quantify the nucleic acid
(DNA/RNA) present in a
sample, during the PCR
reaction is known as a real-
time PCR or q-PCR
 The combination of real-
time PCR (q-PCR) and
reverse transcription PCR
is known as quantitative
RT-PCR or qRT-PCR
https://thumbs.gfycat.com/HarmlessHospitable-size-restricted.gif
Figure 1.3: Real time PCR machine

9. SYBR GREEN
TaqMan Probe
Types of Real time PCR
Probe

10. SYBR Green
 This is a dye that provide
prominent fluorescent signal
when bind at minor groove of
DNA
 Other fluorescent dye like
Ethidium Bromide or Acridine
Orange can also be used but
SYBR Green is better used for
high signal intensity
 Fluoresces only when bound to
double stranded DNA
 Great option for monitoring
amplification of any double-
stranded DNA sequence
https://i.makeagif.com/media/3-08-2016/ggk7ZR.gif
Figure 1.4: Amplification of DNA with SYBR-Green

11. TaqMan probe
https://thumbs.gfycat.com/BrilliantTartBlobfish-size_restricted.gif
 R is reporter fluorophore, w
 hich emits at a wavelength
absorbed by the quencher
fluorophore
 DNA polymerase starts
extending primers moving
towards the probe
 The probe is degraded,
reporter is released from the
quencher and starts to emit
fluorescence
Figure 1.5: Removal of probe and light emitted by reporter

12. Advantages of Real-time PCR
Limitations
 It is time efficient
 Automated, fast and reliable
 It is more sensitive, specific and efficient
 Major advantage is quantification
 Contained less chances of contamination
 High output
 Broad uses
 Instrument itself is too costly as compared with
conventional PCR
 Multiplexing is still limited in Real-time PCR

13. Applications of RT_PCR
 Diagnosing COVID-19
 Amplify small amounts of RNA from specimens into
deoxyribonucleic acid (DNA), which is replicated until
SARS-CoV-2 is detectable if present.
 It quantify absolute amount of virus present in a sample in
rapid time
 Ct value in RT-PCR test detect load of virus
https://pubs.rsc.org/image/article//AY/d1ayh/d1ay00947h-f2_hi-res.gif
Figure 1.7: Molecular diagnostic in the area of COVID-19

14.  Diagnosis of a single gene and multi-genic disease
 Gene expression level in different tissues influenced by the
microRNA
 Recovery in cancer therapy
 Cancer diagnosis
 Microbial load in the fermented sample, soil sample, water
sample, food and food spoilage can be accurately estimated
 Helps to detect the amount of the gene expressed into the
GMO
 Detection and quantification of pathogens
 Used in forensic studies, evolutionary studies, mutation
creation, fossil studies and in other applied fields
Applications of RT_PCR

15. Video: RT-PCR test of corona virus is done

16. References
 https://youtu.be/WKvJ4iBgs7M
 Alvarez, M.L., & Doné, S, C (2014). SYBR® Green and TaqMan
Quantitative PCR Arrays: Expression Profile of Genes Relevant to
a Pathway or a Disease State.
 Arya, M.,Shergill, I. S., Williamson, M., Gommersall, L., Arya, N.,
& Patel, H. R. (2005). Basic-principles-of-real-time/quantitative
PCR.Expert Rev Mol Diagn, 5(2), 209-219.
doi:10.1586/14737159.5.2.209.
 Chien, A.,Edgar, D. B., & Trela, J. M.
(1976).Deoxyribonucleic/acid polymerase from the extreme
thermophile Thermus aquaticus. Journal of Bacteriology127/(3),
1550.
 Huggett, J., Dheda, K., Bustin, S., & Zumla, A. (2005). Real-time
RT-PCR normalisation; strategies and considerations. Genes &
Immunity, 6(4), 279-284.
 Gibson, U. E., Heid, C. A., & Williams, P. M. (1996). A novel
method for real time quantitative RT-PCR. Genome research, 6(10),
995-1001.
 Mo, Y., Wan, R., & Zhang, Q. (2012). Application of reverse
transcription-PCR and real-time PCR in nanotoxicity research.
In Nanotoxicity (pp. 99-112). Humana Press, Totowa, NJ.