Identification of small non coding RNA

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Identification of small non coding RNA

Postby Sultan » Aug 14 2015 10:32 am

Our lab have done small RNA seq to find out the small RNAs of DENV infected mosquitos cells. I have got one small non-coding RNA, that shows similar bands (in northern blot) of the unifected mosquitos cells as well. Can some body suggest what should I do to confirm that it is similar or homolog of the mosquito miRNA. I have checked the miR Base but was not able to find a similar miRNA. Thanks
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Re: Identification of small non coding RNA

Postby mchlbrmn » Aug 14 2015 12:57 pm

I actually haven't worked with miRNAs, but I'll answer from other gene experience in case it might help.
First, you could try to wash the Northern blot more stringently to see if the bands wash off of one sample but not another sample. If you're using a small oligo probe, however, you may have a narrow stringency window, or if the oligo is only part of the target, differences may not be in the region probed.
The other approach if this was a gene would be to PCR, clone if necessary, and sequence the PCR product or clones from different cells. Actually, you may find primers that distinguish two species from each other without sequencing. However, as I said, I don't have miRNA experience and I don't know about potential difficulties in PCR of miRNAs.
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