I'm trying to detect a miRNA on a northern blot without any success. My synthetic miRNA control is working so I think my blotting condition are fine so I'm not sure if I'm loosing my miRNA in the extraction process, RNA transfer or if the corsslinking isn't good enough.
Here is what I've been doing so hopefully someone call give me advice or tell me what I'm doing wrong
I am looking at miRNA16 from HEK293 cells, I have validated the the miRNA is highly expressed via qRTPCR
- RNA extraction: trizol tried with and w/o EtOH wash. I also tried miRvana.
- Transfer: semi-dry transfer to NytranSPC or Brightstar plus in 0.5XTBE 1hr 200mA. The gel has no RNA when I stain post transfer. Is it possible that I am over transferring to the other side/out of the membrane?
- crosslinking: 1-2hr baking at 80C (I do not have a commercial crosslinker)
- Probe: FAM-LNA from Exiqon and then AP anti FAM