RT-PCR
RT-PCR stands for “Reverse Transcriptase - Polymerase Chain Reaction”. In this
procedure, we use Reverse transcriptase to make cDNA, then PCR to amplify a specific gene.
The Reverse transcriptase converts RNA into DNA. It needs a “primer” to do this. The
primer is a short DNA sequence that is complementary to part of the RNA. We will not be
making the second strand of DNA, as we don’t need it for our experiments. To make DNA, the
Reverse transcriptase needs dNTPs and an appropriate salt solution.
After making the cDNA strand, we will use Ribonuclease H (RNase H) to remove the RNA.
The H stands for hybrids, so the RNase digests the RNA of an RNA-DNA hybrid. After the
RNase, we will have single-strands of DNA that are complementary to our mRNA.
Last, we perform PCR on the sample. In this case, we use primers from specific genes.
After the PCR, if we see bands on gel electrophoresis, it suggests that the gene was transcribed
and therefore the mRNA was present in your mRNA sample.
Materials
SuperScript™ First-strand Synthesis System for RT-PCR
Primers for the specific genes you wish to amplify
Methods
Notes: Wear gloves and use autoclaved tips for the first few steps (until the RT step is started)
RT procedure
1) Use the following amounts to prepare samples in 0.2 ml PCR tubes:
Component Sample No RT
control*
Control RNA*
Your mRNA Up to 8 μl Up to 8 μl 0 μl
Control RNA 0 μl 0 μl 1 μl
10 mM dNTP mix 1 μl 1 μl 1 μl
Primer 1 μl 1 μl 0 μl
DEPC-treated water To 10 μl total volume To 10 μl total 8 μl
Notes: The No RT control tests for DNA contamination. The control sample is a positive
control. These controls are often not necessary.
* Only run these samples if instructed to do so.
2) Use the iCycler to incubate the samples at 70°C for 5 minutes, and then place them on ice for
1 minute.
3) Prepare the following reaction mixture. Be sure to add each item in the order that they are
listed. The standard way to do this is to make a “cocktail” of these ingredients, then add an
Last printed on: October 1, 2007 1
