Question: Transcript Specif qPCR primer
0
gravatar for ravihansa82
11 weeks ago by
ravihansa8260
Sri Lanka
ravihansa8260 wrote:

I have a gene which is having 10 transcript variants. However, only 4 transcripts are protein coding ORFs. I need to find out the level of expression of only one transcript out of 4 protein coding transcripts. I used NCBI Primer blast tool to design qPCR primers. however, designed primers not only detect expected transcript but also detect other transcripts variants. during primer designing with Primer BLAST i used these parameters. expected product length 70-200bp, from the Exon/intron selection I selected exon -exon junction.all the other parameters set as default.

I am happy if someone can help me to find out ONLY one transcript variant-specific primer for qPCR out of several other transcripts from the same gene.

gene genome • 123 views
ADD COMMENTlink written 11 weeks ago by ravihansa8260

The target has no unique sequence that can distinguish it from the other transcripts? Take a look at my SOP for designing primers, written a number of years ago, if you need help: Designing a single set of primers and probe for a genomic region of interest.

In essence Primer3 is a good tool: http://primer3.ut.ee/

ADD REPLYlink written 11 weeks ago by Kevin Blighe42k
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