Dear Brinkhof,

When I did the extraction using the mirVana kit enriching to small miRNA step I can't saw any result in qPCR also. So, I entered in contact with thermo scientific's technical support that explained that the enrichment step for small miRNA does not work, to miRNA qPCR technique at least. In my case when I did the extraction until only the total RNA extraction, using the mirVana protocol (using the columns that accompany the kit but without enrichment for small RNA), I could see the miRNA gene expression estimation by qPCR technique.

Did you ever tried this?

@ Pedro Paulo Gattai . Thanks for your answer. Indeed I would like to do qPCR with my miRNA, but first I want to make sure I have miRNA to amplify.

@ Matthias R. Schaefer . After Nanodrop analysis I only did PAGE and Sybr stain. No Northern Blot has been done. I am aware of the minute amounts of miRNAs in the total RNA pool, though still i hoped to be able to visualise this using high cell numbers for extraction. Do you think the bioanalyzer small RNA kit might confirm the presence of miRNA? Or should I just trust miRNAs are present since other small RNA such as tRNAs are detectable? Or is Northern the only way to confirm miRNA presence in the samples?

Thanks for your help.

@ Matthias R. Schaefer. Thank you very much for your advice. Although I have quite some experience with Westerns, it has been ages ago since I did Northern blotting. For the marker I use an NEB microRNA ladder which includes a biotin labelled probe. Could you suggest a protocol to do NB? The only methods I found use horizontal blotting. Since we only have the vertical (western) blotting systems (Mini-Protean; Bio Rad) I wondered if it is possible to use that for Northerns as well? Any other suggestions would be very helpful. Thanks again.