Reverse Transcriptase polymerase chain reaction.

1. RT-PCR
BY- ARGHYA CHOWDHURY (ROLL NO.: 18162027), M.PHARM(1ST YEAR)
Department of Pharmaceutical Engineering and Technology ,IITBHU, Varanasi

2. INTRODUCTION
• RT-PCR is that technology by which RNA molecules are converted into their complementary DNA
(cDNA) sequences by reverse transcriptase, followed by the amplification of the newly synthesized
cDNA by standard PCR procedures.
• Reverse transcription polymerase chain reaction (RT-PCR) is one of many variants of polymerase chain
reaction (PCR).
• This technique is commonly used in molecular biology to detect RNA expression.
• RT-PCR is often confused with real-time polymerase chain reaction (qPCR). However, they are separate
and distinct techniques.
• RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA)
transcripts from RNA.

3. HISTORY
• Howard Temin from the University of Wisconsin–Madison made the discovery of reverse
transcriptase in RSV (Rous Sarcoma Virus) in 1970.
• Which were then later independently isolated by David Baltimore in 1970 from two RNA
tumour viruses: R-MLV (Rauscher- Murine Leukaemia Virus) and RSV (Baltimore, 1970).
• The technique of polymerase chain reaction (PCR) was invented by Kary Banks Mullis in the
year 1983.
• In 1985, a joint venture was established between Cetus Corporation and Perkin-Elmer, another
US based Biotech Company to design thermal cycler instruments and reagents for PCR and in
1987.
• In 1989, Science magazine selected PCR as the major scientific development and Taq DNA
polymerase as the molecule of the year.
• qPCR introduced in 1992 by Higuchi and co-workers (Higuchi et al., 1992) enables detection
of fluorescent reporter dye, such as SYBR Green I to measure the amplification of DNA at
each cycle of PCR.

4. REQUIREMENT IN PCR
• A DNA template that contains the DNA target region to amplify
• A DNA polymerase, an enzyme that polymerizes new DNA strands; heat-resistant Taq polymerase is
especially common, as it is more likely to remain intact during the high-temperature DNA denaturation
process
• Two DNA primers that are complementary to the 3' (three prime) ends of each of the sense and anti-
sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded
region of DNA; without primers there is no double-stranded initiation site at which the polymerase can
bind); specific primers that are complementary to the DNA target region are selected beforehand, and are
often custom-made in a laboratory or purchased from commercial biochemical suppliers
• Deoxynucleoside triphosphates, or dNTPs (sometimes called deoxynucleotide
triphosphates"; nucleotides containing triphosphate groups), the building blocks from which the DNA
polymerase synthesizes a new DNA strand
• A buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA
polymerase
• Bivalent cations, typically magnesium (Mg) or manganese (Mn) ions; Mg2+ is the most common, but
Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn2+ concentration increases the error
rate during DNA synthesis
• Monovalent cations, typically potassium (K) ions.

5. TAQ POLYMERASE
• Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus
aquaticus from which it was originally isolated.
• Thermus aquaticus is a gram negative, rod – shaped bacterium.
• It is resistant upto pH 9.
• T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was
identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required
during PCR.
• Taq's optimum temperature for activity is 75–80 °C, with a half-life of greater than 2 hours at 92.5 °C, 40
minutes at 95 °C and 9 minutes at 97.5 °C, and can replicate a 1000 base pair strand of DNA in less than
10 seconds at 72 °C.

6. PCR

7. WHY RT-PCR
Disadvantage Of traditional PCR
• Poor precision (Northern could even be better)
• Low sensitivity
• Low resolution
• Non-automated
• Results are not expressed as numbers
• Ethidium bromide staining is not very quantitative

8. WHY RT-PCR
Advantages Of Real-Time PCR
• amplification can be monitored real-time.
• wider dynamic range of up to 1010-fold.
• no post-PCR processing of products (No gel-based analysis at the end of the
PCR reaction ⇒ high throughput) .
• ultra-rapid cycling (30 minutes to 2 hours) .
• highly sequence-specific.

9. TYPES OF PCR
Two step RT PCR
• Traditionally, RT-PCR involves two steps: the RT reaction and PCR amplification.
• RNA is first reverse transcribed into complementary DNA ( cDNA) using an enzyme, reverse
transcriptase.
• The resulting cDNA is used as templates for subsequent PCR amplification using primers
specific for one or more genes.
One step RT PCR
• RT-PCR can also be carried out as one-step RT-PCR in which all reaction components are
mixed in one tube prior to initiation of the reaction.
• Although one-step RT-PCR offers simplicity and convenience and minimizes the possibility
for contamination, the resulting cDNA cannot be used for detecting multiple messages from a
single RNA sample as in two-step RT-PCR.

10. ONE STEP RT-PCR PROTOCOL
• Select a one-step RT-PCR kit, which should include a mix with reverse transcriptase and the PCR
system such as Taq DNA Polymerase and a proofreading polymerase.
• Obtain all necessary materials, equipment and instruments (kits should include a detailed list of
necessary items).
• Prepare a reaction mix, which will include dNTPs, primers, template RNA, necessary enzymes and
a buffer solution.
• Add the mix to a PCR tube for each reaction. Then add the template RNA.
• Place PCR tubes in the thermal cycler to begin cycling. The first cycle is reverse transcription to
synthesize cDNA. The second cycle is initial denaturation. During this cycle reverse transcriptase is
inactivated. The next 40 to 50 cycles are the amplification program, which consists of three steps:
(1) denaturation, (2) annealing, (3) elongation.
• The RT-PCR products can then be analyzed with gel electrophoresis.

11. TWO STEP RT-PCR PROTOCOL
• Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This
method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step, use
only intact, high quality RNA for the best results. The primer for two-step does not have to be sequence specific.
Step one
• 1. Combine template RNA, primer, dNTP mix, and nuclease-free water in a PCR tube.
• 2. Add RNase inhibitor and reverse transcriptase to the PCR tube.
• 3. Place PCR tube in thermal cycler for one cycle that includes annealing, extending and then inactivating reverse
transcriptase.
• 4. Proceed directly to PCR or store on ice until PCR can be performed.
Step two
• 1. Add a master mix (containing buffer, dNTP mix, MgCl2, Taq polymerase and nuclease-free water) to each PCR
tube.
• 2. Add appropriate primer.
• 3. Place PCR tubes in thermal cycler for 30 cycles of the amplification program, which includes three steps: (1)
denaturation, (2) annealing, (3) elongation.
• 4. The RT-PCR products can then be analyzed with gel electrophoresis.

12. ONE STEP RT-PCR
• All reaction components are mixed in one tube prior to initiation of the reaction.
• Although one-step RT-PCR offers simplicity and convenience and minimizes the possibility for
contamination, the resulting cDNA cannot be used for detecting multiple messages from a single RNA
sample as in two-step RT-PCR

13. TWO STEP RT-PCR
• Traditionally, RT-PCR involves two steps:
the RT reaction and PCR amplification which
can be simple PCR or qPCR.
• RNA is first reverse transcribed into
complementary DNA ( cDNA ) using an
enzyme, reverse transcriptase.
• The resulting cDNA is used as templates for
subsequent PCR amplification using primers
specific for one or more genes.

14. COMPARISON OF ONE-STEP & TWO-STEP RT-PCR PROCEDURES
Subjects Two-Step Procedure One-Step Procedure
Prime first strand cDNA with: • Oligo(dT) primer
• Random hexamers
• Gene-specific primers
Gene-specific primers
Provides Flexibility
• Choice of primer
• Choice of amplification system
• Ability to save some RNA
sample for later use
• Ability to optimize for difficult
RT-PCR (combine with
Platinum® enzymes for higher
specificity or combine with
Platinum® Pfx for greater
fidelity)
Convenience
• Amplifcation enzymes
premixed with reverse
transcriptase
• Fewer pipetting steps and
reduced chances of
contamination
• High sensitivity
Recommended uses: Ideal for detection or quantifying
several messages from, a single
sample
• Ideal for analysis of large
numbers of samples
• Ideal for real-time quantitative

15. CDNA
• Complementary DNA (cDNA) is DNA synthesized from a single stranded RNA (e.g.,
messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase.
• cDNA is derived from mRNA, so it contains only exons, with no introns.
• cDNA is also produced naturally by retroviruses(such as HIV-1, HIV-2, simian
immunodeficiency virus, etc.) and then integrated into the host's genome.
• Reverse transcriptase PCR (RT-PCR) uses the enzyme reverse transcriptase to make
a cDNA copy of mRNA from an organism and then uses PCR to amplify the cDNA.

16. CDNA SYNTHESIS
• A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
• The same cell processes the pre-mRNA strands by removing introns, and adding a poly-A tail and 5’ Methyl-Guanine
cap (this is known as post-transcriptional modification)
• This mixture of mature mRNA strands is extracted from the cell. The poly-A tail of the post-transcriptional mRNA
can be taken advantage of with oligo(dT) beads in an affinity chromatography assay.
• A poly-T oligonucleotide primer is hybridized onto the poly-A tail of the mature mRNA template, or random
hexamer primers can be added which contain every possible 6 base single strand of DNA and can therefore hybridize
anywhere on the RNA (Reverse transcriptase requires this double-stranded segment as a primer to start its operation.)
• Reverse transcriptase is added, along with deoxynucleotide triphosphates (A, T, G, C). This synthesizes one
complementary strand of DNA hybridized to the original mRNA strand.
• To synthesize an additional DNA strand, traditionally one would digest the RNA of the hybrid strand, using an
enzyme like RNase H, or through alkali digestion method.
• After digestion of the RNA, a single stranded DNA (ssDNA) is left and because single stranded nucleic acids are
hydrophobic, it tends to loop around itself. It is likely that the ssDNA forms a hairpin loop at the 3' end.
• From the hairpin loop, a DNA polymerase can then use it as a primer to transcribe a complementary sequence for the
ss cDNA.
• Now, you should be left with a double stranded cDNA with identical sequence as the mRNA of interest.

17. CRUCIAL ASPECT & CONTROLS OF RT-PCR EXPERIMENT
• DNA contamination must be avoided
• To avoid DNA contamination, two approaches can be used:
1. Treat the sample with DNase (A deoxyribonuclease is any enzyme that catalyzes the
hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA).
2. Use in the reaction a negative control which does not contain reverse transcriptase
enzyme (-RT).
• If the DNA is effectively eliminated from the starting material (RNA sample):
1. Amplification using “-RT” cDNA template should show no product.
2. “+RT” cDNA template should result in a product.

18. REAL-TIME RT-PCR
• The emergence of novel fluorescent DNA labeling techniques in the past few years have
enabled the analysis and detection of PCR products in real-time and has consequently led to
the widespread adoption of real-time RT-PCR for the analysis of gene expression.
• Currently, there are four different fluorescent DNA probes available for the real-time RT-PCR
detection of PCR products: SYBR Green, TaqMan, Molecular Beacons, and Scorpions.
• A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative
polymerase chain reaction (qPCR).

19. SYBR GREEN
• When the SYBR Green binds to the double-
stranded DNA of the PCR products, it will emit
light upon excitation. The intensity of the
fluorescence increases as the PCR products
accumulate.
• This technique is easy to use since designing of
probes is not necessary given lack of specificity
of its binding.
• However, since the dye does not discriminate the
double stranded DNA from the PCR products and
those from the primer-dimers, overestimation of
the target concentration is a common problem.
Where accurate quantification is an absolute
necessity, further assay for the validation of
results must be performed.
• Nevertheless, amongst the real-time RT-PCR
product detection methods, SYBR Green is the
most economical and easiest to use.

20. TAQMAN PROBES
• The two most popular alternatives to SYBR Green
are TaqMan and molecular beacons, both of which
are relying on FRET for quantitation.
• TaqMan probes are oligonucleotides that have a
fluorescent probe attached to the 5' end and a
quencher to the 3' end.
• During PCR amplification, these probes will
hybridize to the target sequences located in the
amplicon and as polymerase replicates the template
with TaqMan bound, it also cleaves the fluorescent
probe due to polymerase 5'- nuclease activity.
• This separates the fluorescent and quenching dyes
and FRET no longer occurs. Fluorescence
increases in each cycle, proportional to the rate of
probe cleavage.

21. MOLECULAR BEACON PROBES
• Similar to the TaqMan probes, Molecular Beacons
also make use of fluorescent probes attached to the
5' end and a quencher attached to the 3' end of an
oligonucleotide substrate, but FRET only occurs
when the quenching dye is directly adjacent to the
fluorescent dye.
• However, whereas the TaqMan fluorescent probes
are cleaved during amplification, Molecular
Beacon probes remain intact and rebind to a new
target during each reaction cycle.
• When free in solution, the close proximity of the
fluorescent probe and the quencher molecule
prevents fluorescence.
• When Molecular Beacon probes hybridize to a
target, the fluorescent dye and the quencher are
separated resulting in the emittance of light upon
excitation.

22. SCORPION PROBES
• The Scorpion probes, like Molecular Beacon, will not
be fluorescent active in an unhybridized state, again,
due to the fluorescent probe on the 5' end being
quenched by the moiety on the 3' end of an
oligonucleotide.
• With Scorpions, the probe and primer present as one
molecule. However, the 3' end contain a primer, the 5'
end contain a hairpin structure of probe and the loop is
complementary to the target.
• A PCR blockers lies between the primer and the probe
prevent the polymerase copying the probe.
• After amplification, heating cause the 3 primers to
unfold, then cooling allowing the complementary
probe to aneal which prevent the hairpin reforming
again and then separate the fluorophore and quencher
releasing fluorescence.

23. REAL TIME PCR INSTRUMENT

24. APPLICATIONS
Research methods
• RT-PCR is commonly used in research methods to measure gene expression. For example, Lin et al.
used qRT-PCR to measure expression of Gal genes in yeast cells. First, Lin et al. engineered a
mutation of a protein suspected to participate in the regulation of Gal genes. This mutation was
hypothesized to selectively abolish Gal expression. To confirm this, gene expression levels of yeast
cells containing this mutation were analyzed using qRT-PCR. The researchers were able to
conclusively determine that the mutation of this regulatory protein reduced Gal expression .
Northern blot analysis is used to study the RNA's gene expression further.
Gene insertion
• RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most
eukaryotic genes contain introns, which are present in the genome but not in the mature mRNA, the
cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of
reverse transcriptase) DNA sequence that would be directly translated into protein after
transcription. When these genes are expressed in prokaryotic cells for the sake of protein production
or purification, the RNA produced directly from transcription need not undergo splicing as the
transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of
eukaryotes).

25. APPLICATIONS
Genetic disease diagnosis
• RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan syndrome. This genetic disease is
caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal uric acid urinary stone
and symptoms similar to gout. Analyzing a pregnant mother and a fetus for mRNA expression levels of
HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–Nyhan
syndrome.
Cancer detection
• Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and
monitor response to therapy. Circulating tumor cells produce unique mRNA transcripts depending on the
type of cancer. The goal is to determine which mRNA transcripts serve as the best biomarkers for a
particular cancer cell type and then analyze its expression levels with RT-PCR.
• RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA,
such as Influenza virus A and retroviruses like HIV.

26. REFERENCES
• Int. J. Adv. Res. Biol.Sci. 1(7): (2014): 65–80
• Fraga, Dean & Meulia, Tea & Fenster, Steven. (2014). Current Protocols Essential Laboratory
Techniques. Current Protocols Essential Laboratory Techniques. 10.3.1-10.3.40
• Xi L, Nicastri DG, El-Hefnawy T, Hughes SJ, Luketich JD, Godfrey TE (July 2007). "Optimal markers for
real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma,
breast, colon, esophageal, head and neck, and lung cancers". Clin. Chem. 53 (7): 1206–15
• Torres RJ, Garcia MG, Puig JG (December 2012). "Carrier and prenatal diagnosis of Lesch-Nyhan disease
due to a defect in HPRT gene expression regulation". Gene. 511 (2): 306–7

27. CONCLUSION
Real-time PCR is a revolutionary technique and is becoming the standard method for quantifying
mRNA levels from organs, cells, or cell cultures. Compared with previously used endpoint PCR
assays, the technique is very fast, accurate, and sensitive, and it has a decreased potential for PCR
contamination. Overall, the technique has enabled scientists to gain a better insight into many
immunological mechanisms and diseases in a fast and relatively automated way.

28. Thanks for listening