RT-PCR primer design - (Aug/05/2013 )
I'm designing some primers for RT-PCR (qPCR) and to do this, I find the mRNA sequence of the gene of interest on Ncbi and Ensembl. Then I copy the sequence from ncbi into Primer3 to select primers and find which exons the primers sit by using Ensembl. The trouble is that I can't always find matching mRNA sequences in Ncbi and Ensembl so I can't always find which exons the primers sit in (which is important to determine if there is genomic contamination). Please can someone offer some guidance?
It works well to just use NCBI gene: http://www.ncbi.nlm.nih.gov/gene database in combination with Primer-Blast: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Search for your gene name, scroll down to see how many mRNA variants (NM_......) are listed. Pick the one that you want to detect by qPCR, then just copy-paste the accession number (NM_....) into the search window of Primer-Blast.
Thank you! I just figured out another way too :)
My two cents: First obtain the refseq number in NCBI, then use Primer-Blast to design primers.
Top Ten Pitfalls in Quantitative Real-time PCR Primer Probe Design and Use
or go to http://www.origene.com
and click on "Register To View Sequence" and write an email even a fake one to see sequence:
Thank you very much for all the links and guidance. Fab ideas. I'm only now worried that the primers I've designed for qPCR will form products that are greater than 150bp in length (up to 240bp but most are ~170bp). Anyway, will see how it goes.
I've recently written a very complete protocol for designing primers for SYBR green qPCR, and posted it on our alternative careers group page:
It even explains how to do clustal alignments and use this information in Primer-Blast in case you want your primers to detect multiple mRNA variants of the same gene that will all give products of the same size. I personally always set my amplicon sizes to be between 90 and 300bp, and this has worked well for me hundreds of times.