Presently, we are trying to isolation miRNA from oocytes. But miRNeasy is not working for isolation of miRNA from a pool of 25 oocytes. Why it is not working with us because we are using low number of oocytes or miRNeasy kit does not work with oocyte type cells.
Assuming you lysed cells/tissue throughly, kits are not sensitive to what type of cells you use.
I think the main problem is that you are using 25 cells (oocytes) which will not yield a lot of RNA.
If you are looking at miRNAs, which are typically also less abundant then mRNA, your abundance (i.e. by Cts) will be even less.
How much RNA are you getting, and your cNDA input- some kits claim amplification for up to 35 cts using 1pg RNA.
Kiessling, thanks for quick reply.
Infact, we are thoroughly pipetting and vortexing as per kit. I guess this must rupture oocytes. Main issue is with quality i.e. 260/280 ratio, which is very low at 1.4 to 1.5. However, yield was almost ok (15ng/ul). Once we got ct at 21 for miRNA using same kit and quality of RNA was excellent in that case.
Perhaps the issue now becomes the abundance of the target template miRNA.
Remember PCR is a doubling process (assuming 100% efficiency), so if there's less template they will amplify later than the ones that have a lot of template (i.e. the one for 21 Cts that you've got).
For example for some highly abundant miRNAs in your oocytes you can achieve Ct of 21, whereas with the ones with low abundance they are not detected. You can try to find some papers that use RNA seq in oocytes to find their abundance and see if this correlates with your Cts.
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