Real Time qPCR - qPCR SYBR greenI primer design (May/12/2005 )
Hi, I would like to know the optimal and maximal distance at which primers have to be designed from 3' -(polyA), for qPCR with SYBR green. May I analyse genes with primers located at different distances form 3' -(polyA)?? It would be nice if someone knows a good qPCR-SYBR green website.
Can you be a little more detailed?
From your question, I am assuming you looking at gene expression by measuring mRNA transcripts.
Are you talking about the gene specific primers for first strand synthesis for the RT-PCR (1 step or 2?)or primers for the cDNA amplification during the SYBR green reaction?
If you are talking first strand synthesis then why not use Oligo (dT) primers since they actually bind to and extend from the poly A tail.
Why is primer distance from poly A tail important? Are you looking at polycistronic mRNA?
Hi, I already have the cDNA (I used oligodT primer); my question is about primers just for the qPCR-SYBR green reaction. I'm analyzing the relative quantitation of gene expression. My gene has a lot of isoforms, but the common region adequate for primer design is as far as 2-3 Kb from polyA tail. I read that optimal distance is about 0,5-1 Kb (related to the RT reaction efficiency), is it really important for primer design? Thanks for your help.
Depends on your rna. If your rna forms a lot of secondary structures and first strand synthesis is performed at low temperatures then the oligodt primers might have had problems extending out to 2-3 kb.
There is one very good website: http://pathmicro.med.sc.edu/pcr/realtime-home.htm
Ch it out. Le me know if u find it useful!
Are you trying to detect and quantify the different isoforms of your mRNA trascript?
qPCR for 2-3kb gene is not recomended. As your amplicon goes longer, your PCR efficiency will decrease propotionally. At the end you can quatify them even though your PCR is work.
Optimum PCR amplicon for qPCR should be 200-300 bp as theory say. However,
I tried 750bp before and the PCR effeciency was about 92%.