RNA-seq doesn't match qPCR and western blot results
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7 weeks ago
heinyxiao • 0

Hello! I really appreciate it if anyone can help me with this issue!

Recently we used siRNA to knock down a gene, and did RNA-seq to find the downstream targets of this gene. We performed qRT-PCR and Western blot to make sure the gene was successfully knocked down before submitting it to sequencing. But when we check the RNA-seq results, this weird thing happened... The expression of this gene was not reduced - both the ctrl and treatment group have ~90 counts of this gene.

I used both STAR and HISAT2 for alignment, followed by DESeq2 normalization, but both of them generated similar results.

Thanks so much if you could provide any suggestions!

qPCR siRNA RNA-seq • 334 views
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After reading your issue, did you considerate that your siRNA could only have post-translational effects? i.e. only affects the protein expression and not the mRNA/transcript stability. Also, it is important to considerate the number of cells that were successfully transfected.

Best regards!

Entering edit mode

Thanks for replying!

I feel the siRNA has knockdown effects on both mRNA and protein level, since we also did qRT-PCR and the results showed ~70% knockdown efficiency... After sequencing, we got back the leftover samples and did qRT-PCR again - the knockdown group still having ~50% less expression on the target gene.

Another issue was, after analyzing the differential expressed genes, we found only 4 genes were significantly affected - which is too few for downstream analysis.

So I was wondering maybe there's something that happened with my analysis...

We did the paired-end 3'-tagged RNA-seq and the read length was 38bp. Do you have suggestions on which aligner to use and what special arguments should I set up?

Many thanks!!!

Entering edit mode

I guess that the length of your reads could affect the alignment. I consider that they are a little bit short, are you interested in a specific population of RNA? Also, I suggest you to check the annotation of aligned/mapped reads by using RSEqC. Follow the read.distribution.py module.

Best regards


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