I am doing RT-PCR for viroid detection using universal primers. In the initial RNA denaturation step, RT-Primer should be used before first strand cDNA synthesis in 2 step RT-PCR. I have not come across the use of RT primer while using universal primers.Can any one help me out?
In your RT, you use hexa nucleotide primers to generate the complementary DNA. These primers are a mix of primers with a random insertion of each nucleotide.
These primers are your RT primers and are universal. They must be added in the mix of RT.
The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.
Hi There
yes RT PCR is so called because you make a copy of your RNA by reverse transcription during RT - that is your cDNA - and then subsequently you amplify the cDNA made from your RNA using specific primers by PCR
Here are some useful links describing the initial RT reaction:
http://www.lifetechnologies.com/uk/en/home/life-science/pcr/reverse-transcription/rna-priming-strategies.html
http://www.thermoscientificbio.com/applications/basic-rt-qpcr/
I have specific RT protocols/SOPs I can send you if you wish but google will provide you with everything you need
Let me know if you would like me to provide SOPs
Good luck and best wishes !!
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