which exon to choose when designing primers for RT-PCR? - splicing isoforms are different between NCBI and Genecard (Jun/26/2011 )
hi guys, I have a question when designing primers for RT-PCR. I want to check the endogenous level of one gene in different cell lines. To design the primers, I need to get forward primer in one exon and the reverse in another neighboring exon. But one gene may have several splicing isoforms and some isoforms may not have the exons that I choose. In this case, two sets of primers which have different target regions may give quite different results. Then how should I get a meaningful result?
another question is that gene information on splicing isoforms provided by NCBI and Genecard is different. Which one should I believe?
Thanks in advance.
Ideally, you should design your primers on the exons shared by most if not all isoforms or splicing variants. You can use UCSC genome browser which gives you better visualization of gene structures.
thank you guys, I will try those tools.