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Necessity of strand-specific RT-qPCR to validate RNA-seq hits?
Hi all,
I'm very new to all of this NGS stuff and I am concerned that my ignorance on a particular issue is going to be debilitating...again.
I have a large set of RNAseq data where I am looking for differential intron retention events between my two experimental conditions. The bioinformatic analysis has revealed several interesting hits, so I would think my next step would be to validate those hits using RT-qPCR. I have designed primers on each end of the retained intron (5' exon->intron -- intron->exon 3') and performed one-step RT-qPCR.
My issue is with someone I've been working with that is telling me that I need be doing all of my validation using tagged, strand-specific RT primers because of the possibility of non-specific antisense transcription. I understand that some of the hits we get will overlap closely with some other genes, but I am curious if this is truly something that I need to be concerned about. The majority of hits I have looked at thus far do not overlap with neighboring genes on the opposite strand, so I don't understand why he is pushing this issue. I have been scouring the www for papers or information regarding this and have come up empty--which was another a red flag for me. I expected to at least come across something in methods sections in papers but google, bing, and pubmed are giving me nothing. I don't know who else to ask. If anyone has any experience in this issue and would be willing to help me--I would be seriously grateful. Thanks in advance.
I'm very new to all of this NGS stuff and I am concerned that my ignorance on a particular issue is going to be debilitating...again.
I have a large set of RNAseq data where I am looking for differential intron retention events between my two experimental conditions. The bioinformatic analysis has revealed several interesting hits, so I would think my next step would be to validate those hits using RT-qPCR. I have designed primers on each end of the retained intron (5' exon->intron -- intron->exon 3') and performed one-step RT-qPCR.
My issue is with someone I've been working with that is telling me that I need be doing all of my validation using tagged, strand-specific RT primers because of the possibility of non-specific antisense transcription. I understand that some of the hits we get will overlap closely with some other genes, but I am curious if this is truly something that I need to be concerned about. The majority of hits I have looked at thus far do not overlap with neighboring genes on the opposite strand, so I don't understand why he is pushing this issue. I have been scouring the www for papers or information regarding this and have come up empty--which was another a red flag for me. I expected to at least come across something in methods sections in papers but google, bing, and pubmed are giving me nothing. I don't know who else to ask. If anyone has any experience in this issue and would be willing to help me--I would be seriously grateful. Thanks in advance.