rna isolation from bone marrow cells - (Aug/15/2006 )
I need to isolate total RNA from samples stored with RNA later with 3 million of Bone Marrow cells each.
Protocol is the standar trizol.
I have done the extraction and I just get 10-20ng/microl. and a low 260/280 ratio ( 1.6-1.7).
I have got some doubts:
1. are 3 million cells enough or should I pellet a higher number of cells?
2. Should I wash the rna later from the sample before the isolation or is it not necessary?
3. Anyone knows if storing the cells with rna later is a good idea? or could someone recommend me a better method? (because I think I lose cells when I wash the sample to remove the rna later)
Someone kind enough to help me please........
well RNA later is not supposed to be washed out before the RNA extraction and this product is a good idea if you don't extract iummediately the RNA. I prefer extract the RNAs quick, and eventually store them in ethanol for up to 1y at -20 (supposed, but i've done only 2month storage) and finish the whole procedure when having all samples.
A low RNA ratio is not surprising when concentration of RNA is low in the cuvette.
I've read sthg on the effect of solvent on 260/280 ratio and posted it in attached picture.
Maybe use of water or DEPC water (wich after autoclave contains slight traces of ethanol (DPC degradation product)) increases the OD230 and thus decreases the ratio of 230 as well