Download Reverse transcription-pcr (rt-pcr)

Reverse
transcription-pcr
(rt-pcr)
Dr. Hani Alhadrami
[email protected]
www.hanialhadrami.kau.edu.sa
Overview !  
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Several techniques are available to detect
and analyse RNA. Examples of these techniques are: Northern
blots, Western blots, and RNase protection
assay (RPA). Theses techniques are labour-intensive and
requires large amount of fully intact RNA. Overview !  
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In contrast, newer method based on
PCR is less time-consuming, more
sensitive and is able to tolerate partially
degraded RNA samples. This method is called: Reverse
Trasncription PCR
(RT-PCR)
Invention of RT-PCR
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The idea of RT-PCR is based
upon retroviruses. Retroviruses have an RNA
rather than DNA genome. So they must use reverse
transcriptase (RT) to covert
their RNA genomes into DNA. Human Immunodeficiency Virus
(HIV) is an example of a
retrovirus. Application of Rt-PCR
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RT-PCR is used to test RNA
extracts from different tissues for
the presence of a particular
transcript in order to determine
the expression pattern of a gene. Principle of Rt-PCR
RT-PCR enables RNA to be used as
a template for PCR. !   RT-PCR comprises two steps: 1.  The first step is reverse
transcription (also called cDNA
synthesis). 2.  The second step is the utilisation of
cDNA as a template in PCR. !  
Steps of Rt-PCR
1.  cDNA synthesis:
This is the process of
converting RNA to DNA
using an enzyme called
reverse transcriptase. The resulting DNA is
complementary to the RNA
template, so it is called
complementary DNA
(cDNA). !  
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Steps of Rt-PCR
2.  The use of cDNA as a template in PCR:
Steps of Rt-PCR
2.  The use of cDNA as a template
in PCR:
Once the cDNA is synthesised from
step-1, the PCR primers and Taq
polymerase are added and the
experiment proceeds exactly as in the
standard PCR technique.
The amount of PCR product is
indicative of the starting amount of
input cDNA (and by interference, RNA
that it was generated from). !  
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Steps of Rt-PCR
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RT-PCR can be performed in two
steps, with an RT reaction occurring
first, then using that product as a
template in a PCR reaction. Alternatively, RT-PCR can be
carried out in one step with both
reaction occurring in one tube. Ingredients of Rt-PCR !  
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The reaction tube of RT-PCR contains the following: Reverse transcriptase: the enzyme catalyses the reverse
transcription reaction. There are several commercially
available enzymes usually isolated from retroviruses. Buffer.!
dNTPs (like those used in PCR), and they will be
incorporated into the newly synthesised cDNA strand. Primers: which are required to synthesis cDNA. RTPCR requires only one primer because only one strand
of cDNA is made. Primers categories Primers fall into three categories: Oligo-dT, randomers
(hexamers) and gene specific. 1.  Oligo-dT primers: will specifically anneal to polyA tails found on
most eukaryotic mRNAs. This type of primers cannot be used
with prokaryotic RNA. 2.  Gene Specific primers: cannot be used if we want to prime cDNA
synthesis from all the RNAs in the cell.
3.  Random hexamers primers: have the ability to anneal to all types
of RNA without knowledge of sequence. They are a pool of
primers designed to represent all possible combinations of sixbase pair stretches. This design allows for the primers to bind to
all RNA sequences. !  
Crucial Aspect Controls RT-PCR experiment !  
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DNA contamination must be avoid
in RT-PCR experiment. If DNA still present in the RNA
sample, it can be amplified by the
gene specific primers that amplify
the cDNA leading to inaccurate
quantification of mRNA levels. Crucial Aspect Controls RT-PCR experiment To avoid DNA contamination, two approaches
can be used: 1.  Treat the sample with DNase. 2.  Use in the reaction a negative control which dose
not contain reverse transcriptase enzyme (-RT). If the DNA is effectively eliminated from the
starting material (RNA sample), amplification
using -RT cDNA template should show no
product. The corresponding +RT cDNA
template should result in a product. !  
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THANK YOU!!
REFERENCE:
MOLECULAR BIOLOGY TECHNIQUES: A
CLASSROOM LABORATORY MAUAL