Question: Please advise on primer designing for qRT-PCR analysis of Trinity transcripts
gravatar for seta
19 months ago by
seta920 wrote:

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.

qrt-pcr primer design trinity • 623 views
ADD COMMENTlink modified 19 months ago by Biogeek240 • written 19 months ago by seta920
gravatar for Biogeek
19 months ago by
Biogeek240 wrote:

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.

ADD COMMENTlink written 19 months ago by Biogeek240
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1454 users visited in the last hour