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7.1 years ago
simplitia
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130
Hi all, so I'm doing a DGE analysis shRNA knockdown vs GFP. However I notice that when I plot cpm count for the gene in question is still highly expressed when compared to GFP control. qPCR prior shows a clean knockdown. I'm wondering if this due to the shRNA breaking down the mature mRNA and leaving fragments behind? Is this why a something like EdgeR normalization would still pick up count for the gene? Is there a way I can measure how well the knockdown? In other words I'm expecting low to zero counts for the gene that has been shRNA treatment, but I'm not seeing it and I'm wondering if this has do with picking up the fragments?
thanks. A.
If the qPCR sample and the RNAseq sample come from different experiments, it's always possible that one of them failed. As usual with experiments, it is always a good idea to do multiple replicates. Also if you do not limit your RNAseq to poly-A RNAs, you may be counting remaining shRNA sequences.
Does the shRNA cut in between the sequence that the qPCR primers amplify? If so, that explains the qPCR result. As for the RNA-seq, I would guess that the cut is rather towards the 5', leaving a major chunk of the mRNA, including the polyA intact. Is the RNA polyA-enriched or ribo-depleted?
Hi @ATpoint. I am facing similar kind of issue. The gene which showed 40% of silencing in protein level, however doesn't show significant difference when performing differential expression. We used ribo depletion kit.
I checked for the isoform percentage provided by the rsem, for each known transcript. It showed that maximum reads were coming from the transcript that's canonical and not from an isoform that is yet to be discovered or other reported isoforms
Please open a new question, adding details and code. A PCA plot is a good diagnostic, please add that, too.
Hi @ATpoint.
I have opened a new question here:TERF2 shRNA silencing is visible in WB and qPCR but not reflected in PE RNASeq
please have a look.