I did not come across any rt primer when universal primers are used for viroid detection. But in some protocols especially 2 step rt pcr they have mentioned rt primer in RNA denaturation step before first strand cDNA synthesis. can anyone help me out?
just understand the concept of Reverse Transcriptase PCR i.e., a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.
Hello again
before you carry out RT PCR to detect your gene of interest by RT PCR it might be useful to do some basic reading about the concept of RT PCR
In brief, as mentioned in my prior answer RT converts RNA to cDNA and the cDNA then acts as a substrate for primer specific PCR. Hence RT PCR rather than just PCR. When you make cDNA from RNA in a reverse transcription (RT) reaction you make multiple copies of your RNA as cDNA. That basically means you have much more material for your gene specific PCR increasing the chances of success. It is possible to PCR directly from RNA but is very tricky, unusual and invariably unreliable
When you reverse transcribe you parent RNA into cDNA you add one of two univeral primers: Oligo dT which primes the poly A tail of mRNA and/or random hexamers which random prime the entire mRNA coding and non coding sequence (ORF) and increase the efficiciency of RT compared to oligo dT alone (especially if your gene specific primers are near the 5' end of your gene and especially when the mRNA is more than 1kb in length)
Having reverse transcribed with tthese universal primers you then subject your RT reaction to a heat inactivation step to kill the enzyme. This heat inactivated reaction mix is then used in gene specific PCR
Here are some useful links as per my previous e mail
http://www.lifetechnologies.com/uk/en/home/life-science/pcr/reverse-transcription/rna-priming-strategies.html
http://www.thermoscientificbio.com/applications/basic-rt-qpcr/
If you would like me to provide further clarification or specific protocols, drop me a line
Best wishes !!
For viroid detection using oligo dT primer will not work as viroids don't have a poly A region. You can use random hexamers but don't usually work well for viroids due to their extensive secondary structure. For viroid detection you have two choices. So for viroid detection you will have to use gene specific primer in your RT step. It will probably be much easier to do 1 step RT-PCR (that is set up your PCR reaction with RT added and have 50 degree 30 minute cycle before the PCR reaction)
For viroids I normally use the following
30 min @ 50 degrees followed by a 3min @ 94 degrees then 30-40 cycles of 94 for 30 sec, 50-60 degrees 30 sec and 72 degrees for 30-60 seconds.
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