I need to experimentally validate the novel microRNAs in my patient cohorts. Before that is it necessary to do cloning of mature/precursor mir sequence followed by sanger. If so , Can anyone share me the protocol ? or their any alternative strategy ? please suggest ?
Hi Gurjeet,
cloning should take you huge efforts and money, why not simply block your miRNA expression by siRNA and see if the expression is really blocked (and therefore reapply miRNAseq and mirdeep2 analysis). it's just an idea, never done.
fred
Hi Gurjeet,
I would suggest performing Northern Blots of your novel miRNAs to see if they are really there. I haven't done this myself, but look for miRNA papers from Gunter Meister's lab at the University of Regensburg, Germany.
My PhD was all about miRNA. Why did not you perform northern blotting to see if the size distribution of your product ?
Thanks a lot everyone. Mohamed Samir , northern blot was my first option for charaterization of novel microRNAs.But i think radioactive probe was used in northern blot , so its very difficult to obtain ethical clearance for radioactive probe . Can you suggest/send me alternative protocol for characterization of novel microRNAs ? .thanks
I would suggest using qRT-PCR to confirm expression of novel microRNAs after bioinformatic analysis. There are many available Kits for this purpose. I used All-in-one™ miRNA qRT-PCR detection kit (GeneCopoeia,
Rockville, MD). It's easy and straightforward protocol.
http://www.genecopoeia.com/wp-content/uploads/2016/03/All-in-One-miRNA-qRT-PCR-Detection-Kit-Manual-2016New-Cat.no_.pdf
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