mRNA isolation without DNA!

Csaba Kiss csakis at ki.se
Tue Feb 8 04:12:42 EST 2000


Hi guys!
I would like to do subtractive hybridization on cDNA. I want to isolate
mRNA, which is free from even traces of DNA. The acidic phenol method is the
generally used method to isolate total RNA, and it is based on the fact that
the DNA will remain in the  phenolic phase if the phenol's pH is acidic (ph
5.0 ?). Then people do mRNA separation on oligo-T beads or columns.  I have
a feeling that these separations do not get rid of your residual DNA
totally. There is another way of isolating cytoplasmic RNA. You can gently
lyse the cells in a way that the nuclei remain intact. You pellet down the
nuclei, and work with the supernatant. This way you got rid of all the
genomic DNA. However, in such a mild lysis buffer there is no Rnase
inhibitor. How would I prevent my mRNA being degraded by the endogenous
Rnase? Dynal recommends to work quickly on ice. I have a gut feeling that
this does not eliminate RNA degradation. What do you think?

Csaba
csakis at ki.se






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