I put in each well 1 million cells, and the cells looks good and I use zymogen kit, but I get a low concentration from the RNA around 20-30 ng/micro liter.
Hi Manal,
Did u try mechanical destruction of cell membrane or u just make the enzymatic lysis. I strongly recommend to make mechanical destruction by reuptake cells for about 15 times by a syringe. Have a nice experiment.
Hi Manal,
In addition to what has been suggested above, I typically use a bioanalyser (you can use northern blots aswell) to determine the RNA integrity (based on 28S and 18S ribosomal RNA). This will tell you whether your RNA is degraded. RNA degradation is a very common problem during RNA isolation. Hope that helps.
cells should be washed 3-4 times with PBS because even traces of cellular medium lowers RNA yield and quality.
Also, such concentration even from 1 million cells should be enough for RT and qPCR. My co-worker spent 1 year on efficient RNA isolation from NK cells - and after 13 (!) tested kits she gave up - the quality was perfect (RINs 9-10) but the yield was 10-50 ng/ul max.
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