mRNA of knockout still detectable by qPCR?

We have deleted a section of gDNA from upstream of the start codon to mid-way through a gene (ie all that's left at the gDNA level is the final 2 exons of the gene and it lacks the canonical start codon). If we target the deleted region for qPCR we see nothing (ie undetermined Ct values), but if we target the final 2 exons we still see low relative mRNA expression (0.1-0.4 relative to housekeeping). Is this normal/expected? Are other folks still detecting small amounts of mRNA by qPCR following CRISPR-induced nonsense mutations and partial gene deletions? I would hope/assume this mRNA is on its way to being degraded by NMD, but I worry it could also possibly be circumventing NMD and be translated using a noncanonical start codon to form a small truncated protein (we're lacking antibodies for western at the moment).


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Pearl Lamiaalmost 3 years ago

We do not yet understand all of the nuances to NMD. But it is possible you have not met the “50-55 nucleotide rule” defined by Lynne Maquat’s lab. On the other hand, and consistent with what you see, we have generated a truncated transcript that easily fulfills this rule and yet we still see mRNA!

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