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4.7 years ago
rob.costa1234
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How well single-cell RNAseq correlate with qRTPCR. In my case gene was not significantly detected in SCRNAseq but when we performed qRTPCR on the whole RNA same cells, we can see its high expression. Is it possible?
Yes, this can happen. For a more elaborate answer please add details. Which gene, how high is it expressed relative to the control and what are the control genes you normalized qPCR against? Which scRNA-seq protocol, plate- or droplet-based?
MYC gene, IN 10X based ScRNA-seq I see >2 read counts in around 10 cells in tSNE plot For QRTPCR we took same cells extracted total RNA and did qRT PCR normalized against actin it is 50 FC Will the SCRTPCR be the better option to cross-check SCRNASeq?
Depending on the number of total cells (10 out of 50 is a lot different from 10 out of 5000), coverage, etc, dropout may be playing a role. When you say 50 FC, you mean it's got a fold change of 50 as compared to actin?
Yes FC is against actin. Drop out means filtered out in ScRNA seq?
There are a small number of genes that for whatever reasons, are just not detected as highly by sparse scRNA-seq platforms (like 10x) as they are expressed in reality. It's possible your gene falls into this category, but it's difficult to say without looking at the data as a whole.
rob.costa1234 : Please consider validating answers/comments you have received for your past questions. That is an appropriate way of acknowledging help you receive here.
Sure I Will do the needful
Certainly possible, there is a big difference in the sensitivity between the two techniques. The amount of starting material is vastly different so I wouldn't be surprised. One thing you could do is to check the expression level at the "cluster level" and not at single cell level, the assumption being cells within a given cluster have same/similar transcriptomes.