(RNA Template-Specific PCR, RS-PCR)

Biotechnology-Theory Lec. 4 3rd Student-Medical Analysis 2021

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REVERSE TRANSCRIPTASE PCR (RT-PCR)

(RNA Template-Specific PCR, RS-PCR)

Sensitive method for the detection and analysis of rare mRNA transcripts or

other RNAs present in low abundance are an important aspect of most cell/

molecular biology studies. RNA cannot serve as a template for PCR, so it must

first be reverse transcribed into DNA known as the" complementary DNA or

cDNA." Powell et al. first described a combined technique, now commonly

known as RT-PCR in which reverse transcription (RT) is coupled with PCR

amplification of the resulting cDNA.

Reverse transcription polymerase chain reaction (RT-PCR): is one of

many variants of polymerase chain reaction (PCR). This technique is commonly

used in biotechnology qualitatively to detect gene expression through creation of

complementary DNA (cDNA) transcripts from RNA. It is an extraordinarily

sensitive method to detect as few as l-100 copies of a specific RNA. This is also

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.

Analysis of gene expression requires accurate determination of mRNA levels.

But PCR is based on amplification of DNA rather than RNA, so how can it be

used for mRNA analysis? The answer is that first, mRNA is converted into DNA

using the well-known process of reverse transcription, which is used by RNA

viruses to convert their genomic RNA into a DNA within the host cell; and

second, PCR amplification is performed on the resulting complementary DNA

(cDNA).


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Amplification of mRNA molecules to study gene expression can be achieved

by a method that combines two sequential enzymatic steps: the synthesis of DNA

from the RNA template using dNTPs by an RNA-dependent DNA polymerase

[reverse transcriptase] (RTase enzyme) derived by AMV (Avian Myoblastosis

Virus) reverse transcriptase or Thermus thermophilus (Tth) DNA polymerase with

using Oligo dT primer or random hexamer primer and followed by PCR using of

a heat stable DNA polymerase (Taq polymerase). The extremely high sensitivity

of RT-PCR enables us to detect rare mRNAs, mRNAs in small numbers of cells

or in small amounts of tissue as well as mRNAs expressed in mixed-cell

populations.


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This enzyme is a RNA dependent DNA polymerase able to perform the

following reactions:

ο Synthesizes a DNA strand on an RNA template.

ο Removes the RNA strand from the DNA: RNA duplex (RNase H activity).

ο Synthesizes a second DNA strand on the DNA template.

This enzyme is used in the replication cycle of Retroviruses, a group of viruses

that converts their RNA genomes into DNA that will then integrate in the genomes

of host cells. The enzyme preparations that are commercially available are

therefore typically of retroviral origin, the most common of which are avian

myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia

virus (M-MuLV) reverse transcriptase. Tth polymerase, an enzyme isolated from

the bacterium Thermus thermophilus, is a heats table DNA polymerase.

The AMV and m-MuLV viral RTases are highly processive and are able to

synthesize cDNAs of up to 10 kb. Tth DNA Polymerase is able to synthesize

cDNA in the range of 1.0 - 2.0 kb, which is sufficient since fragments of <1 kb

are usually used for PCRs. The unique advantage of the Tth DNA Polymerase is

its ability to perform both reverse transcription and PCR amplification in a one-

step reaction.


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RT PCR is a two-step process:

1) In the first step of RT-PCR, called the "first strand reaction,"

complementary DNA is made from a messenger RNA template using dNTPs

and an RNA-dependent DNA polymerase (reverse transcriptase) through the

process of reverse transcription. RT-PCR exploits a characteristic of mature

mRNAs known as the 3' polyadenylated region, commonly called the poly (A)

tail, as a common binding site for poly (T) DNA primers. In the case of

bacterial mRNA, which lack a poly (A) tail sequence-specific primers can be

generated to amplify the target mRNA sequence. These primers will anneal to

the 3' end of every mRNA in the solution, allowing 5'-3' synthesis of

complementary DNA by the reverse transcriptase enzyme. Complementary

DNA can also be prepared from mRNA by using gene specific primer or

random hexamer primers.

2) After the reverse transcriptase reaction is complete, and complementary DNA

has been generated from the original single stranded, standard polymerase

chain reaction, termed the "second strand reaction," is initiated.

After approximately 30 cycles, millions of copies of the sequence of

interest are generated. The original RNA template is degraded by RNase H,

leaving pure cDNA (Plus spare primers).


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I. Two step RT-PCR. The synthesis of cDNA is performed with reverse

transcriptase from AMV, moleney murine leukemia virus (M-MuLV), or

Thermus thermophilus (Tth) DNA polymerase in the first step, followed by

PCR with an appropriate thermostable DNA polymerase. The two-step

reaction requires that the reaction tube is opened after cDNA synthesis and

reagents are added for the PCR part of the procedure. This is inconvenient and

increases the risk of contamination.

II. One-step RT -PCR. The cDNA synthesis and the PCR are performed together

in a single tube. Two techniques are in use for running one-step RT-PCRs. The

first technique uses Tth DNA Polymerase for carrying reverse transcription and

PCR reactions. The second uses AMV reverse transcriptase and Taq DNA

Polymerase.


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There are three types of primers that may be used for reverse transcription:

1. Oligo (dT) [deoxy-thymine nucleotides] 12-18 primer. This primer binds to the

endogenous poly (A) +tail at the 3’-end of mammalian mRNA. A reaction with this

primer frequently produces a full-length cDNA product.

2. Random Hexanucleotide Primers. These primers can bind to mRNA templates at

any complementary site and will give partial length (short) cDNAs. These primers

may be better for overcoming the difficulties caused by template secondary structure.

The random primers may also transcribe more 5' - regions of the RNA.

3. Specific Oligonucleotide Primers. These primers can be used for selectively

priming the RNA of interest. This approach has been used very successfully in

diagnostic assays, as well as in basic research.


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RT-PCR has found significant application in the field of disease diagnostics

and some of these applications in the field of infectious diseases.

ѽ Many clinically important viruses have genomes composed of RNA, RT-PCR

is useful for detecting such viruses. RT-PCR has also been used for the

detection of the viral causes of meningitis and meningoencephalitis, such as

enteroviruses and the West Nile virus. RT-PCR is being used for the detection

of the following viruses: Dengue virus, Hantavirus, Human metapneumovirus,

Severe acute respiratory syndrome (SARS) and Coronavirus (COVID-19).

ѽ Quantitative RT-PCR assays are commonly used for the detection of HIV and

HCV viral load (amount of these viruses present in the blood of a patient)

testing.

ѽ RT-PCR may also be used to detect other microorganisms (bacteria, parasites,

and fungi) by targeting their rRNA. This approach is better than detection of

DNA, as the presence of RNA is more likely associated with the presence of

viable organisms.

ѽ Detection of mRNA using RT-PCR helps to study the gene expression of both

microorganisms and human host cells.

(RNA Template-Specific PCR, RS-PCR)

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