Please advise on primer designing for qRT-PCR analysis of Trinity transcripts

Question

[seta]

Hi all,

I have to examine the expression profile of some selected genes (transcript) generated from a de novo transcriptome assembly of a non-model plant by Trinity tool in the various samples via qRT-PCR analysis. As you know, the output of Trinity is multiple transcript isoforms of the same gene. Since I want to assay the expression profile in the several samples, which have not been already sequenced and analyzed, I have no idea which isoform will be differentially expressed during qRT-PCR analysis. So, I am not sure which isoform should be considered for primer designing. I also thought about designing primer pairs from similar regions of all isoforms, but I still cannot make decision what I should do. Could you please help me on this issue?

Your hep would be highly appreciated.

Answer 1

[Biogeek]

If you design primers for similar regions across all isoforms. Perhaps you will end up with multiple products ( peaks) in your qPCR which is not what you want. You want a clean single peak on melting.

Why don't you go with the longest isoform? This is why I much prefer working at the gene level. I usually follow trinity up with cd hit eat and cap3 for clustering and re-alignment.

I think your best bet is to order a few primers designed around the longest isoform. You want your product to be about 100-150bp in size.