miRNA Identification - (Jan/28/2016 )

Hi,

I am completely novice to miRNA field. Just started reading and getting information. Can you expert please answer my questions?

How do you make sure that miRNA exists in your treatment samples? For example, you isolate miRNA using the kits available in the market. How will you identify which miRNA is that ? If it is first of its kind?
I read about qPCR for miRNA but there also you need forward primer which should be specific to your miRNA, how will you do thatn when you don't know which miRNA is that.

There are so many questions, can somebody share some links where the basic questions are answered?

Please experts help me!

Thanks

-neuron-

Here are a few resources.

You can find many miRNA sequences in miRBase, which helps for designing primers.

http://www.mirbase.org/

You can probe for an miRNA in situ.

Lagendijk AK, Moulton JD, Bakkers J. Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biol Open. 2012 Jun 15;1(6):566-9. doi: 10.1242/bio.2012810. Epub 2012 Apr 11.

http://bio.biologists.org/content/early/2012/04/11/bio.2012810

-Jon Moulton-

Thanks for the links Jon Moulton! Is there any simple way to probe miRNA in lab? We can't do in situ hybridization. I thought we can do denaturing urea PAGE after isolating miRNA using commercially available kits. But I could not find a single paper wherein the gel picture is published. I don't know if miRNA can be seen on PAGE as they are so small. Any suggestion will be a great help for me.

Thanks

-neuron-

Northern blotting can work. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 2007;5(8): e203. http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0050203

There are commonly used PCR methods; hopefully someone more familiar with them can help out.

-Jon Moulton-

You can design specific primer to analysis the quantification of miRNA. And also, SDS-PAGE could be used to run miRNA, too, you just need to add urea in it. You can search protocol on google or papers.

http://www.ncbi.nlm.nih.gov/pubmed/22808238

-Andrea Fortina-