Why do I get a positive gene expression with qPCR for one my genes but in with when doing bulk RNA seq I get a count of 0?

Question

[bioinfo]

Hello,

I have done qPCR on my samples for a gene of interest and I normally get a CT value of around 25. However, I ended up doing bulk RNA seq and I get a count of 0 across all my samples. I used an illumina sequencer, bcl2fastq for demultiplexing and kallisto for mapping.

I am not sure why that is happening and I would appreciate any suggestions/help.

Thank you

Answer 1

[ksh4718]

Possibly it's DNA contamination in your qPCR. Do you have exon-exon junction spanning primers? What do the primer meltcurves show (if using SYBR green technology)? If you load your aligned reads on a browser do you see reads for that gene from the RNAseq? If so, the mapping / counting has errors.

[dsull]

For pseudoalignment methods (like kallisto), it can't be a counting error (the EM algorithm will resolve multimapping transcripts; a count of 0 means that it just wasn't mapped). Pseudoalignment is one of the most sensitive mapping methods so if you see zero counts, I'd say it's most likely that your transcript of interest isn't present in your cDNA library.

Really hard to diagnose since we don't know your primer sequences, your qPCR protocol, your library prep protocol, your sequencing protocol, or even the commands/files you're giving to kallisto.