Hi, everyone. I recently analyzed a set of RNA-seq data. Based on the fold change rank, a batch of ncRNA shows as top up-regulated in RNA-seq data. I am new to RNA-seq analysis. Is it normal in RNA-seq data? Can I conclude that the ncRNAs are specific to my experiment after verified by RT-PCR? If so, what's the normal ways of analyse a ncRNA function? Like prediction of binding to mRNA/DNA, prediction of secondary structure, over-expression, konckout. Is knockdown works well? Thank you so much for help.
Hi Lei,
Well, first of all, it is important to check which kit was used for library preparation. The most popular kit types for RNA-seq at the moment include mRNA (when you capture only poliA-containing mRNA) and Whole Transcriptome (when you catch all RNA`s of particular size range) sequencing. It immediately implies that if you have RNA-seq from poliA-based method, you do not have ncRNA`s in your library (though it is speculated that some ncRNA`s have poliA-tail, you still can not claim anything about them). In this case you should filter your data to use only protein-coding genes in the differential expression analysis. If you have RNA-seq data from the Whole Transcriptome analysis, you already can include non-protein-coding transcripts into your analysis. Anyway, you should be careful not to include microRNA`s, for example, because they are too small for the usual WT kits and require special treatment from the beginning of the library prep.
Also, if you want to do RT-PCR on ncRNA`es, you should use the random priming for RT instead of poliA primers.
By the way, you should check the p-values (or FDR, which is more suitable for RNA-seq), not only the log-fold-change. Could be that the variation between replicates is so high, that you exclude such genes anyway.
Overexpression and Knockout in-vivo work fine (from the technical aspect I mean, you acquire/lose the expression of the particular transcript).
Knockdown should also work, though I never did it by my own. But in principle, I do not see factors which should prevent RNA-interference.
Best,
Michail
Thanks a lot, Dr. Michail Yekelchyk. Since the experiment is done by previous post-doc, who left the lab 2 years ago. I do not have the information of the methods/chip used for this experiment. Is there a way to know by checking the number of genes detected or some specific RNA sequence only shown in Whole Transcriptome sequencing, for C. elegans data?
After the cutoff by FDR and fold change, still a lot of ncRNAs shown as top-regulated. I think I need to do some realtime PCR.
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