‫كلية التقنية الطبية صرمان‬

‫قسم التقنيات الحيوية والهندسة الوراثية الطبية‬

Reverse Transcription
Polymerase Chain Reaction
Produced by :Takwa Morad Shlaki
 introduction
REVERSE TRANSCRIPTION–POLYMERASE CHAIN
REACTION
Reverse transcription polymerase chain reaction (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). It is primarily used to measure the amount of a
specific RNA. This is achieved by monitoring the amplification reaction using
fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined
RT-PCR and qPCR are routinely used for analysis of gene expression and quantification
.of viral RNA in research and clinical settings

RT-PCR uses RNA as starting material for in vitro nucleic acid amplification. The
discovery of retroviral reverse transcriptase in the early 1970s ultimately made RT-PCR
possible. Reverse transcriptase is an RNA-dependent DNA polymerase, catalyzing
DNA synthesis using RNA as the template. The end product is known as
complementary DNA (cDNA). cDNA is not subject to RNase degradation, making it
more stable than RNA. In RT-PCR, the starting RNA is subsequently degraded, dsDNA
is produced, and PCR amplification proceeds in the usual manner.

RNA extraction kits for both manual and automated RNA purification exist and, when
combined with RT-PCR, make RNA analysis in the clinical laboratory virtually as rapid
and equally sensitive as PCR-based DNA amplification.

RT-PCR is commonly used in the diagnosis and quantification of RNA virus infections
(e.g., human immunodeficiency virus and hepatitis C virus) and the analysis of mRNA
transcripts such as those produced by translocations commonly associated with non-
Hodgkin's lymphomas, leukemias, and sarcomas.

Gene expression profiling is likely to have a major impact on molecular

diagnostics in the coming years and will depend on RNA analysis using RT-PCR
and possibly high-density arrays.
Protocol of RT-PCR

RT-PCR can be carried out by the one-step RT-PCR protocol or the two-step RT-
PCR protocol.
 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.

1) One-step RT-PCR take mRNA targets (up to 6 kb) and subjects them to reverse
transcription and then PCR amplification in a single test tube. Use only intact, high
quality RNA for the best results. Be sure to use a sequence-specific primer.

2) 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.

3) Obtain all necessary materials, equipment and instruments (kits should include a
detailed list of necessary items).

4) Prepare a reaction mix, which will include dNTPs, primers, template RNA, necessary
enzymes and a buffer solution.
5) Add the mix to a PCR tube for each reaction. Then add the template RNA.

6) 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.

7) The RT-PCR products can then be analyzed with gel electrophoresis.

Two step RT-PCR

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.

Advantage of RT-PCR
1) it does not require post PCR processing,.
2) a wide range (>107-fold) of RNA abundance can be measured.
3) it provides insight into both qualitative and quantitative data.
4) Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of
applications( used as diagnostic tools for detecting infectious agents such as the
avian flu virus and SARS-CoV-2).
5) RT PCR has since displaced northern blot as the method of choice for RNA detection
and quantification.
6) Made it theoretically possible to detect the transcripts of practically any gene.
7) Enabled sample amplification and eliminated the need for abundant starting
material required when using northern blot analysis.
8) Provided tolerance for RNA degradation as long as the RNA spanning the primer
is intact.

Challenge of RT-PCR

1) The exponential growth of the reverse transcribed complementary DNA (cDNA)

during the multiple cycles of PCR produces inaccurate end point quantification due
to the difficulty in maintaining linearity.
2) In order to provide accurate detection and quantification of RNA content in a
sample, qRT-PCR was developed using fluorescence-based modification to
monitor the amplification products during each cycle of PCR.
3) The extreme sensitivity of the technique can be a double edged sword since even
the slightest DNA contamination can lead to undesirable results.
4) Additionally, planning and design of quantification studies can be technically
challenging due to the existence of numerous sources of variation including
template concentration and amplification efficiency.
5) Expensive and time consuming.

Applications
The exponential amplification via reverse transcription polymerase chain reaction
provides for a highly sensitive technique in which a very low copy number of RNA
molecules can be detected. RT-PCR is widely used in the diagnosis of genetic
diseases and, semi quantitatively, in the determination of the abundance of specific
different RNA molecules within a cell or tissue as a measure of gene expression
in :

 Research methods
 Gene insertion
 cancer detection
 RNA extracts
 Genetic disease diagnosis
 studying the genomes of viruses, such as (Influenza virus A, retroviruses like
HIV and SARS-CoV-2).
 Relative and absolute quantification of gene expression
 Validation of DNA microarray results
 Variation analysis including SNP discovery