mRNA-based tissue typing: Which transcripts to choose for primer engineering?

Question

[milesdavis]

Hi there,

I am designing PCR primers to identify human tissues and body fluids via differential mRNA expression. Typically there are multiple known transcripts for each target gene. For example, the blood marker ALAS2 has eight protein-coding transcripts in the Gencode transcriptome and three NCBI RefSeq transcripts. Out of these, NCBI chooses a RefSeq Select transcript (here, NM_000032.5 for ALAS2), which is picked based on multiple criteria such as clinical importance and transcript expression.

Ideally, I would try to design primers targeting all of the known transcripts, but this task becomes increasingly difficult in case of more than two or three transcripts, given that one of the primers should be placed on an exon-exon-junction to avoid amplification of gDNA. However, I believe that not all of the transcripts are equally important. Some transcripts might be generally expressed at low levels. So the question here is, if it's worth it to try to target all of the known transcripts.

Which transcript(s) would you use for primer design?

Answer 1

[GouthamAtla]

You could check transcript level expression from GTEx for all tissues ? Exon level expression across tissues is also available but it needs to be used with caution as 3' exons tend to have higher coverage. Gtex also provides junction counts per samples. You could also chose based on junctions that are most expressed per gene. Gencode also provides ORF annotations, if you want further evidence of protein coding exons. I am not a wet-lab biologist so I can only suggest from data perspective.

[milesdavis]

Thanks a lot, GTEx is indeed a very useful ressource. Transcript TPMs and junction read counts are what I was looking for. Great!