Since our RNA is less abundant, can we use the libraries prepared for RNA-seq to validate (qPCR)the results of RNA-seq.
RNA Sequence is the most useful way to explore gene expression profiling as it can be run on Galaxy Online and after mapping and with reference alignment, you can even explore differential gene expression. The helpful tool is https://usegalaxy.org/u/chmy/w/rna-seq-differential-analysis
RNA Sequence is the most useful way to explore gene expression profiling as it can be run on Galaxy Online and after mapping and with reference alignment, you can even explore differential gene expression. The helpful tool is https://usegalaxy.org/u/chmy/w/rna-seq-differential-analysis
Hi Swathi,
You mean doing the PCR on the NGS Libraries, not on the extracted RNA?
You could try but it would be a VERY tricky experiment, depending also on the library you used. Illumina (well done) RNAseq libraries preparation usually fragment RNA in 250-300 bp fragments so you'll need to design your primers inside a read or having the Forward and the reverse on two paired reads (if your sequencings are paired-end). Also consider that your fragments have the adapters attached.
Overall, a qPCR could maybe work if well designed, starting of a couple of reads and knowing the fragment size of your library. But If you have any other choice (total RNA samples) it would be better.
I agree with Umberto, validating your RNA-seq result with qPCR is better done on the original RNA sample. You could do it on your final libraries, but you have to be very careful on designing your primers.
Thank you @umberto palatini and @xinkun Wang.
Since our RNA is highly fragmented and less abundant, we tried various amplification procedures but it is not working well (abundance not matching with RNA-seq results). Whereas with the Library prepared for RNA-seq the Ct is consistent and matching with the rna seq results... So wanted to know whether it could be used to validate my RNA-seq results?
The purpose of the qPCR experiment in this context is to validate RNASeq results using an independent method, so using the RNASeq library itself will just replicate any potential bias or false positive, defeating the point.
Depending on your study, consider i) repeating the experiment to obtain more RNA for qPCR, or ii) using an alternative readout or method to validate the RNASeq finding e.g. addressing protein product level.
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