This presentation talks on the mechanism of Reverse Transcription as well as RT-PCR. This presentation can be used to clearly understand the essential mechanism of RT-PCR, which has been gaining major analytical relevance. This presentation clearly elucidates the steps, advantages, inhibitors, required materials and disadvantages of RT-PCR in a simple and concise manner.

1. Aman Nanavaty
REVERSE
TRANSCRIPTION
AND RT-PCR

2. INDEX
PAGE NUMBER TITLE
1 WHAT IS REVERSE TRANSCRIPTION?
2-3 RETROVIRUSES
4-5
MECHANISM OF REVERSE
TRANSCRIPTION IN RETROVIRUSES
6-7 REVERSE TRANSCRIPTASE
8-9
MECHANISM OF REVERSE
TRANSCRIPTION IN TELOMERES
10 TELOMERASE REVERSE TRANSCRIPTASE
11-12 REVERSE TRANSCRIPTION INHIBITORS
13
SIGNIFICANCE OF REVERSE
TRANSCRIPTION
14-15
REVERSE TRANSCRIPTION POLYMERASE
CHAIN REACTION
16-17 STEPS INVOLVED IN RT-PCR
18 TYPES OF RT-PCR
19 ONE STEP RT-PCR
20-21 TWO STEP RT-PCR
22-23 REAL TIME RT-PCR [qPCR]
24 END POINT RT-PCR
25 RT-PCR APPLICATIONS
26 FACTORS AFFECTING RT-PCR
27 ADVANTAGES OF RT-PCR
28 DISADVANTAGES OF RT-PCR
29 REFERENCES

3. WHAT IS REVERSE
TRANSCRIPTION?
Reverse Transcription (RT) is the process through which retroviral DNA
is synthesised from RNA via Reverse Transcriptase. In simple terms, it
is the synthesis of DNA from a RNA template. It occurs in Retroviruses,
Bacterial Retrons, Telomeres and Retrotransposons.
Reverse Transcription is used by retroviruses to infect cells by
incorporating their genomes into host genomes and by eukaryotes to
extend telomere length via telomerase. A common misconception is that
reverse transcription only occurs in viruses, but actually it occurs in all
eukaryotes and even some bacteria. Eukaryotes use Telomerase to
replenish the telomere cap, thereby countering the Hay
fl
ick Limit.
Retrotransposon mobile elements facilitate the replication of viral
genomes in infected host cells, allowing for the widespread transcription
and translation of the viral genome. Unlike normal transcription, it
involves RNA-dependent DNA Polymerase activity as well as DNA-
dependent DNA polymerase activity.
Ever since its discovery, RT has been extensively studied. RT is used in
RT-PCR that is used for the ampli
fi
cation of both DNA and RNA samples
as well as in pathological studies of RNA viruses.
1

4. RETROVIRUSES
Retroviruses are class VI ssRNA-RT viruses that insert a DNA copy of
its own RNA genome into the host cell’s DNA genome by the process of
Reverse Transcription. Upon invading the host cell, retroviruses use an
enzyme called Reverse Transcriptase that synthesises DNA from a RNA
template. They then utilise another enzyme, Integrase to incorporate the
newly synthesised DNA into the host genome. After the retroviral DNA
has been incorporated into the host genome, the encoded viral genes
are transcribed and translated alongside the host’s genes.
There are 3 major subfamilies of retroviruses:
Oncoretrovirinae- Tumour causing viruses such as HTLV, MLV,
Hepatitis C Virus, RSV
Spumaretrovirinae- Foamy viruses (benign) that do not cause
disease in animals, such as Simian Foamy Virus
Lentivirus- Responsible for chronic diseases with long incubation
times in animals, such as HIV, BIV, Puma Lentivirus and EIAV
Retroviruses have a diameter of around 0.1 um. They have 2 identical
single-stranded RNA molecules that are 7-10 kb long, which form a
kissing-stem loop structure. The RNA is enclosed by a protective lipid
bilayer. Retroviruses encode the following proteins:
Protease (pro)- plays a role in proteolytic cleavage during virion
maturation
Pol- involved in synthesis and integration of viral DNA
Env- involved in initial association and entry of virions into host cell
gag- main components of capsid, consists of MA and NC domains.
Also regulates assembly
These viruses have gained the attention of the scienti
fi
c community due
to their role in many notable diseases such as HIV/AIDS, Avian
Myeloblastosis, Hepatitis C and Human T-cell Leukaemia.
2

5. STRUCTURE OF RETROVIRUSES
RETROVIRUS CLASSIFICATION
3

6. MECHANISM OF REVERSE
TRANSCRIPTION IN
RETROVIRUSES
Retrotranscription consists of the following steps:
1. Lysyl tRNA, acting as primer, hybridises to Primer Binding Site
(PBS) on the viral RNA genome
2. Reverse Transcriptase adds nucleotides that are
complementary to U5 and R regions to the 3’ end of primer- cDNA
synthesis
3. RNase H degrades U5 and R regions on 5’ end
4. tRNA jumps to 3’ end of viral genome, newly synthesised DNA
strands hybridises to complementary R region
5. cDNA extended by nucleotide addition
6. Except for PP sequence, entire viral RNA degraded by RNase
H
7. Using PP sequence as primer, second strand of DNA
synthesised
8. tRNA primer leaves, causing a jump that results in PBS of
second strand to hybridise with complementary PBS of
fi
rst
strand. Formation of dsDNA by strand transfer
9. Both strands extended to form dsDNA copy of viral RNA
genome.
10. dsDNA copy integrated into host genome via Integrase
4

7. PROCESS OF REVERSE TRANSCRIPTION IN RETROVIRUSES
5

8. REVERSE
TRANSCRIPTASE
Reverse Transcriptase is a RNA-dependant DNA polymerase that was
fi
rst isolated from retroviruses. It is a multifunctional enzyme that has
RNA-dependant DNAP, DNA-dependant DNAP and Integrase activity.
Initially believed to be exclusive to retroviruses such as HIV, BIV and
Hepatitis B virus, many different kinds of reverse transcriptases have
been found in eukaryotes, such as humans, Schizosaccharomyces
pombe and Bombyx mori.
The structure of RT varies among species, with HIV-1 RT being a
heterodimer while M-MLV RT is a single 75kDa monomer. AMV RT
consists of 2 subunits, 63 kDa and 95 kDa.
HIV-1 Reverse Transcriptase (HIV-RT) is an asymmetric heterodimer
consisting of 1 p66 subunit and 1 p51 subunit. The p66 subunit is 66kDa
catalytic region that is responsible for the RT activity. On the other hand,
p51 subunit is a 51 kDa protein that supports the p66 subunit. Both
subunits are derived from the PR- catalysed cleavage of Gag-Pol
polyprotein.
RT also has ribonuclease H (RNase H) domain that degrades the RNA
template upon cDNA synthesis. Integrase domain may either be present
or free of RT. Integrase facilitates the entry of synthesised dsDNA copy
of viral RNA into the host cell genome.
RT has a right-hand structure that comprises of
fi
ngers (1-85, 118-155),
palm regions (86-117, 156- 237), thumb region (237-318) and
connection region (319-426); quite similar to most viral nucleic acids
polymerases.
The nucleic-aid binding cleft is formed by p66
fi
ngers, palm, thumb,
connection and p66 RNase H domain. p51 subdomains (connection and
thumb) form the the
fl
oor of the binding cleft.
6

9. REVERSE TRANSCRIPTASE ACTION
7
HIV REVERSE TRANSCRIPTASE STRUCTURE

10. MECHANISM OF REVERSE
TRANSCRIPTION IN
TELOMERES
Telomeres are repetitive DNA sequences present on the ends of the
chromosome which shorten after every subsequent replication. To
reverse the shortening of telomere cap, telomerase catalyses the
addition of CERN (in humans, TTAGGG) to the ends of the
chromosome. This nucleic acid sequence is repeated about 100-1000
times.
The enzyme telomerase attaches to the end of the chromosome. The
enzyme consists of 2 subparts, the catalytic TERT protein and RNA
template-containing TR component.
First, telomerase adds bases complementary to RNA template to the 3’
end of the overhang. Once the lagging strand is suf
fi
ciently elongated,
DNA Polymerase along with Primase use it as a template to synthesise
cDNA to the ends of the chromosome, thereby extending the length of
the telomere cap.
Telomerase acts on most telomeres without regarding the telomere
length, focussing on extending the telomere cap that is shortened on
replication. This process prevents ageing and senescence of old cells,
while bypassing the Hay
fl
ick limit. Telomerase confers immortalisation of
cells by ensuring that multiple replications do not harm the telomere,
that in turn protects the DNA in the chromosome.
8

11. 9
REGULATION OF
TELOMERASE
REVERSE
TRANSCRIPTASE
TELOMERE
REPLICATION IN
EUKARYOTES

12. TELOMERASE REVERSE
TRANSCRIPTASE
The Telomerase enzyme is involved in lengthening the telomeres in
DNA strands, facilitating cells to exceed the Hay
fl
ick limit and achieve
immortalisation. Its main component, Telomerase Reverse Transcriptase
(TERT) is the catalytic subunit that catalyses the addition of nucleotides
(TTAGGG seq) to the ends of chromosome’s telomeres.
It is a ribonucleoprotein polymerase that is up-regulated in rapidly
dividing cells such as embryonic stem cells. TERT confers self-renewal
of stem cells to ensure bypassing of the Hay
fl
ick limit. The pluripotency
and multipotency of stem cells is indicated by high levels of TERT.
The core human telomerase enzyme consists of 2 main components:
1. TERT Protein- comprises of Reverse Transcriptase Domain (RT),
C Terminal extension (CTE), Telomerase Binding Domain (TBRD)
and essential N-Terminal Domain (TEN). TERT palm contains
aspartic acid residues and TERT
fi
ngers bind the incoming
nucleotides. It contains 1132 Amino Acids and has a molecular
weight of 127 kDa.
2. TR Component- comprises of CR4/5, Psuedoknot (contains RNA
template) and H/ACA regions
10
CORE HUMAN
TELOMERASE ENZYME

13. REVERSE
TRANSCRIPTION
INHIBITORS
Telomerase reverse transcriptase has been shown to be inhibited by
catechin derivatives, phytochemicals and nucleoside analogues. Some
phytochemicals such as Genistein and Circumin have been shown to
inhibit telomerase activity by inhibition of mTOR pathway (down
regulation of phosphorylation). Derivatives of Retinoic Acid, Nucleoside
analogues, Quinilone antibiotics and catechin derivatives are being
studied as potential drugs to combat malignancy.
Telomerase is also inhibited by well-known Nucleoside Reverse
Transcriptase Inhibitors (NRTIs) such as abacavir, tenofovir, zidovudine
and stavudine. These NRTIs have demonstrated good potential in vitro,
raising the possibility of 100% effective anti-HIV drugs in the future. An
area of concern is the potentially lethal side effects of abacavir and
stavudine in HIV patients. NRTIs and NtRTIs inhibit RT by competing
with natural deoxynucleotides for incorporation into the growing viral
DNA chain.
Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as
nevirapine and delavirdine are also effective in inhibiting HIV RT. They
work by inducing allosteric changes in retrovirus RT, thereby blocking
the process of reverse transcription. Some studies suggest that
Telomerase RT is unaffected by NNRTIs. NNRTIs inhibit RT by directly
binding to the reverse transcriptase (non-competitive inhibition).
Reverse Transcription is also inhibited by drugs that target integration of
viral DNA copy, known as Integrase Inhibitors. Some examples of
Integrase Inhibitors are bictegravir and elvitegravir.
Despite these drugs’ good inhibitory action on RT, retroviruses are able
to develop resistance to them in a fairly quick time, rendering the drugs
useless after a few generations. This developed resistance has proved
to be a stumbling block for treatment of Hepatitis C and HIV/AIDS.
11

14. 12
MAJOR REVERSE TRANSCRIPTASE INHIBITORS
NRTI MECHANISM

15. SIGNIFICANCE OF
REVERSE
TRANSCRIPTION
•Next-gen sequencing of cDNA
• RT-PCR: RT-PCR [Reverse Transcription - Polymerase Chain
Reaction] is an ef
fi
cient technique used to amplify even minute
quantities of DNA/RNA samples. It is widely used in metagenomics,
metabolomics, ecological diversity studies and species identi
fi
cation.
• Study of Retrovirus Pathology: To understand exact mechanisms of
infection, scientists are studying process of Reverse Transcription in
different retroviruses.
• cDNA labelling: Labelling of cDNA with
fl
uorescent marker (GUS,
GFP, SyBr Green I) is used to elucidate the complete pathway of
genetic info transfer.
• Study of role of Telomerase in Tumours: Recent studies have
shown that unregulated telomerase activity correlates to cancer/
malignancy. Researchers are currently studying the effects of mutation
of TERT gene on normal function.
• In vitro drug studies: The ef
fi
cacy of RT inhibitors such as NRTIs
and Integrase Inhibitors are tested in vitro.
13

16. REVERSE TRANSCRIPTION-
POLYMERASE CHAIN REACTION
Reverse Transcription - Polymerase Chain Reaction (RT-PCR) is an
advanced technique used for the ampli
fi
cation of RNA samples. It combines
Reverse Transcriptase with the ampli
fi
cation of PCR to facilitate better and
accurate ampli
fi
cation of DNA and RNA samples.
RT-PCR produces DNA complementary to the RNA sample (cDNA) and then
using Polymerase Chain Reaction, the cDNA is exponentially ampli
fi
ed for
RNA quanti
fi
cation. Reverse Transcriptase catalyses the generation of cDNA
from RNA sample and Taq Polymerase is involved in polymerising new DNA
strands. The RNA sample serves as template for Reverse Transcriptase. The
Taq Polymerase also requires suitable DNA primers, dNTPS, buffers and
certain bivalent cations along with the DNA template (cDNA).
In modern times, RT-PCR has displaced the time-consuming and inef
fi
cient
method of Northern Blot for RNA quanti
fi
cation. It has allowed for high
ampli
fi
cation of RNA samples without the need of large sample or post-
technique processing. The transcripts of any gene can be practically
analysed by RT-PCR.
RT-PCR is widely used in many streams, such as Food Process Industries,
Cancer Research, Quality Assurance, Drug Design, Pathological Studies,
Environmental Monitoring and Genome Modi
fi
cation
14

17. 15
COVID-19 SCREENING FACILITATED BY RT-PCR

18. 16
STEPS INVOLVED IN RT-PCR
1. Primer Design: Formulate and synthesize highly gene-
speci
fi
c primers with a length 20-30 nucleotides. The primers
must not have a secondary structure, which can interfere in
ampli
fi
cation
2. RNA Extraction: Isolate RNA from sample (environmental,
plant, prokaryotic or animal) using suitable technique
3. Reverse Transcription: Perform Reverse Transcription
using Reverse Transcriptase to generate cDNA from RNA
sample that serves as template
4. PCR: Implement suitable method of PCR (either one step /
two step or real time / end point) and suitable
fl
uorescent
probe/dye to amplify cDNA
5. Analysis of Results: Using sophisticated computer
programs and softwares, generate graphical analysis of
results to determine concentration of RNA sample

19. 17
STEPS INVOLVED IN RT-PCR
SINGLE THERMAL
CYCLE OF RT-PCR

20. 18
REVERSE
TRANSCRIPTASE
POLYMERASE
CHAIN
REACTION
ONE STEP RT-PCR TWO STEP RT-PCR
REAL TIME RT-PCR END POINT RT-PCR

21. 19
ONE STEP RT-PCR
One Step RT-PCR is a popular method of RT-PCR in which both
Reverse Transcription and cDNA ampli
fi
cation via PCR are carried
out in the same test tube. It is a fast and accurate method widely
used in COVID diagnostics, high throughput screening and analysis
of multiple samples.
By carrying out all of the enzymatic reactions in a single test tube, it
eliminates the risk of contamination while ensuring better
optimisation than two step RT-PCR. Lesser handling corresponds to
less time and less variation in results. One step RT-PCR requires
only sequence speci
fi
c primers and is carried out in conditions that
support both reverse transcription and polymerase chain reaction
activity. Results from one step RT-PCR are more reproducible and
accurate.
The limitations of one step RT-PCR include its lack of
fl
exibility,
lower sensitivity due to combined reactions and inability to detect
multiple genes within a single RNA sample. Another drawback is
that it is impossible to optimise the 2 reactions separately.

22. 20
TWO STEP RT-PCR
Two Step RT-PCR uses 2 test tubes, one for Reverse Transcriptase
action and another for RNA ampli
fi
cation. Unlike One Step RT-PCR, it
requires both random nonspeci
fi
c and sequence-speci
fi
c primers. It is
used in cDNA archiving and Multiplex RNA target detection.
Reverse Transcription that generates cDNA using RNA as template is
carried out in the
fi
rst test tube. The cDNA is then transferred to
another test tube followed by addition of sequence-speci
fi
c primer.
PCR is carried out to amplify the sample. Due to the transferring of
cDNA, the risk of contamination is high and the process is more time
consuming than one step RT-PCR. Increased sample handling may
inadvertently introduce contamination that skews results, increasing
the variability of results.
However, two step RT-PCR is well-suited for analysis of multiple
genes within a single RNA sample. The separation of 2 reactions
allow for enhanced priming
fl
exibility,
fi
delity and optimisation of each
step, resulting in ef
fi
cient results. Another advantage is the lack of any
multiplexing. Two step RT-PCR is more
fl
exible and broad-ranged
than one step RT-PCR, although its longer time and intensive
procedure limit its usage.

23. 21

24. 22
REAL TIME RT-PCR [qPCR]
Real Time RT-PCR is considered as the gold standard for the detection of
RNA viruses (SARS-Cov-2, Zika). It is the most preferred mode of RT-PCR,
often used in quanti
fi
cation and array analysis.
It involves analysis and detection of PCR products in real time using suitable
probes. These probes either bind to the DNA or couple to a quencher moiety
via FRET to produce a detectable
fl
uorescent signal. The probes also confer
higher sensitivity as the instrument will only detect the PCR product if the
probes attach. Real Time RT-PCR is more accurate as it measures
quanti
fi
cation at Exponential phase and continuously measures
fl
uorescence
after every cycle of PCR.
Some of the major probes used are:
TaqMan Probes- Modi
fi
ed oligonucleotides with a quencher at 3’ End
and Fluorescent Probe at 5’ End. TaqMan probes are cleaved during
ampli
fi
cation. They are highly speci
fi
c and generate
fl
uorescence by
FRET coupling. Using TaqMan probes is expensive and challenging.
Scorpion Probes- Scorpion extension on 3’ End binds to
complement on amplicon which opens the scorpion structure. This
prevents FRET and
fl
uorescent signal is then measured.
Molecular beacons- Unlike TaqMan probes, they are not cleaved
during ampli
fi
cation. When beacon hybridises to target, dye and
quencher separate which emits light. They are expensive to
synthesize.
Compared to End Point RT-PCR, it has higher sensitivity and speci
fi
city while
also involving lesser steps. These factors make Real Time PCR the RNA
quanti
fi
cation technique of choice.

25. 23

26. 24
END POINT RT-PCR
End Point RT-PCR utilises
fl
uorescent dyes to detect gene expression levels,
rather than
fl
uorescent primers. PCR products obtained after process are
stained with a suitable
fl
uorescent dye, the resulting
fl
uorescence is studied
to determine expression levels.
Commonly used
fl
uorescent dyes include Ethidium Bromide (carcinogenic)
and SyBr green. Alternatively, scintillation counting and Phosphorimager P32
labelling can be used to detect expression. Unlike Real Time RT-PCR, it only
measures the
fl
uorescence after the completion of PCR, resulting in less
accurate results. In this process RNA quanti
fi
cation is performed ad Lag
phase, similar to other traditional techniques. It uses densitometric
techniques to quantify visible PCR products.
There are 3 methods of End Point RT-PCR, namely:
Relative RT-PCR: It is the relative quanti
fi
cation of PCR products
with co-ampli
fi
cation of a suitable internal control along with the gene
of interest. The control is used to normalise the sample. Results
expressed as ratio of gene signal to control signal.
Comparative RT-PCR: Target RNA must compete with ampli
fi
cation
reagents in a single reaction along with an internal standard/control.
The results obtained are then compared with an external RNA
standard curve to determine RNA concentration. Most convenient
method as it does not need investigator.
Competitive RT-PCR: Similar to Comparative RT-PCR in which
standard curve is directly generated from the results obtained after
PCR. It is used for absolute ampli
fi
cation and involves a synthetic
competitor sequence followed by co-ampli
fi
cation.

27. 25
RT-PCR APPLICATIONS
RT-PCR is one of the most widely used laboratory techniques, having
other inef
fi
cient and older methods such Northern Blot and regular PCR.
Methods such as qPCR and Real Time PCR have been used in
quanti
fi
cation and ampli
fi
cation of various RNA samples collected from
key sources.
RT-PCR is being used in detection of cancer as well as in monitoring
patient response to therapy. It is used to detect the transcripts produced
by the circulating tumour cells. Scientists use RT-PCR to determine the
expression levels of these transcripts in cancer patients.
Scientists utilise RT-PCR in gene insertion techniques to insert
eukaryotic genes into prokaryotic genomes. Genes from eukaryotes,
such as plants or insects are inserted into bacterial genomes (such as
Escherichia coli). RT-PCR mediated gene insertion has been used in
genetic engineering modi
fi
cations.
Detection of genetic diseases such as Lesch-Nyhan Syndrome by
analysing mRNA expression levels of mutated HPRT1 is done by RT-
PCR.
Gene expression studies often involve RT-PCR techniques. For
example, expression of target genes involved in important cellular
processes are done using RT-PCR followed by Northern Blot. Gal genes
in yeast were studied using this method.
RT-PCR has been used in detection of contaminant microbes like molds
and yeasts in dairy products (milk, yogurt, etc.) and other foods meant
for human consumption. It is a key technique in Quality Assurance
programs of Food Industries
Genomes of RNA viruses such as HIV, SARS-Cov-2, West Nile Virus,
Zika Virus and In
fl
uenza A virus are studied using extensive RT-PCR.

28. FACTORS AFFECTING
RT-PCR
RT-PCR is affected by:
1. Viral Load of Sample: Results are highly dependant on the
Viral Load of the sample.
2. Collection Procedure: The sample collection may in
fl
uence the
PCR products and results. Before performing RT-PCR, the
collection method should be chosen carefully in accordance to
nature of RNA sample.
3. Nature of Sample: Many times samples from different sources
(but same location/person) can have different results. For
example, in COVID testing, the results from Upper Respiratory
Tract and Lower Respiratory Tract differ.
4. Unsafe transport of PCR products may introduce
contamination
5. Storage: Incorrect storage of reagents involved or PCR
products can lead to false and erroneous results. After this, the
entire process must be repeated, wasting money and time.
6. Primer Bias: Primer Bias due to primer-binding site mismatch
can undermine hybridisation, reducing accuracy and
correctness of results. Primers must be carefully selected before
initiating RT-PCR.
7. Incorrect Interpretation: Sometimes, the results may be
wrongly interpreted from the graph/readings by the researcher.
RT-PCR should be repeated a few times to minimise variation
and ensure accurate and precise results.
8. Varying Probe Dimerisation in different Sample Test Kits
26

29. 27
ADV
ANTAGES OF RT-PCR
1. Due to its high speci
fi
city and sensitivity, RT-PCR can
give a reliable and accurate diagnosis in just a few hours
2. High con
fi
dence detection of low copy targets is possible
3. Compared to other diagnostic methods, it requires less
time
4. Entire process is carried out in a single test tube
5. Unlike standard PCR, does not need post-analysis gel
electrophoresis
6. Lower potential for contamination and hence, less
chance of errors
7. Very wide dynamic range as RT-PCR can detect
between 1-1011 copy targets
8. Provides both quantitative and qualitative information
9. Tolerates sample RNA degradation as long as primer is
intact
10. Easier automation than conventional PCR

30. DISADV
ANTAGES OF RT-PCR
28
1. Negative RT-PCR tests do not always exclude infection
and hence RT-PCR cannot be used as a sole diagnosis tool
2. Requires complex and careful experiment design
3. Higher costs as RT-PCR must be performed by well
trained, experienced professionals in clean and secure labs
4. More time-consuming than ELISA
5. Less scalability due to extensive requirements
6. Short window for detection, as it can only detect presence
of viral genetic material during infection
7. More intensive than conventional PCR techniques
8. RT-PCR doesn’t indicate past infection and whether
patient has developed immunity. Hence, it is not useful in
determining development and spread of the virus

31. REFERENCES
https://www.britannica.com/science/reverse-transcriptase
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881421/
https://en.wikipedia.org/wiki/Reverse_transcriptase
https://www.ncbi.nlm.nih.gov/books/NBK19424/
https://www.yourgenome.org/facts/what-is-a-telomere/
https://en.wikipedia.org/wiki/Telomere
https://pubmed.ncbi.nlm.nih.gov/23166583/
https://www.sciencedirect.com/topics/neuroscience/reverse-
transcriptase#
https://www.creative-biogene.com/support/rt-pcr-protocol.html
https://en.wikipedia.org/wiki/Telomerase_reverse_transcriptase
https://help.medicinalgenomics.com/en/qpcr-vs-end-point-pcr
https://www.ncbi.nlm.nih.gov/books/NBK551504/
https://www.sciencedirect.com/topics/neuroscience/telomerase-
reverse-transcriptase
https://bio.libretexts.org/@go/page/13294
https://phadkelabs.com/blog/advantages-and-limitations-of-real-
time-reverse-transcription-polymerase-chain-reaction-real-time-rt-pcr/
https://en.m.wikipedia.org/wiki/
Reverse_transcription_polymerase_chain_reaction
https://www.thermo
fi
sher.com/in/en/home/brands/thermo-scienti
fi
c/
molecular-biology/molecular-biology-learning-center/molecular-
biology-resource-library/spotlight-articles/onestep-vs-twostep-
rtpcr.html
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189409/
29

32. THANK
YOU