I performed a Mi-seq experiments using cancer cell lines, and as a result a large number of different microRNA showed different modulation levels. Afterthat, based on these data I chose some miRNA to validate using a qPCR and the same sample. But now, all the modulation is invert, like in mi-seq the miR-X was modulated -4 in qPCR is +4. So I don't know what happened in this case if the sequencing or the analysis data were wrong. by the way How have I proceed now? Has anyone ever experienced this?
Hello Tatiana,
As a first step I would double check if in differential expression analysis cases and controls had been properly set. A common error is to switch them by mistake.
Tatiana Takahasi Komoto
In your question not mentioned about miR sequencing reads and qPCR ct value and how normalized gene expression.
So here I am sharing some information about qPCR (ct) vs miR NGS(reads).
In qPCR(ct values)
Decreased ct value gives increased gene expression
Increased ct value gives decreased gene expression
In NGS(count/million miRNA read)
low mean read gives increased gene expression(DCT-Delta CT)
high mean read gives decreased gene expression (DCT)
qPCR(delta ct ) vs miR NGS read
below 0 50428
0-4 30414
0-9 1485
10-14 955
15-19 435
reference
Adopted from Lee et al., 2014
Article A detailed analysis of next generation sequencing reads of m...
Best wishes
Sincerely
Prabu
Thank you, I'm checking bioinformatics analysis and perfoming again qPCR. As a normalized gene I used RNU6B. Maybe this control is not appropriate.
Thank you.
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