We've been doing RNA isolations using the guanidium thiocyanate method of
Chomchynski for years without problems. We have recently had a problem in
our isolations of losing the 28S band, although the 18S band and the tRNA look
fine. We've done duplicate isolations in different cell lines using the same
reagents, and everything looks fine in all the cell lines except the HeLa's,
therefore we've concluded that all our reagents are free of RNases.
We've repeated the isolation twice and the only common factors in the preps in
which 28S disappears is 1) it only occurs in the suspension culture lysates
and 2) during the first phenol/chloroform extraction there is no phase
separation, and we get a white precipitate. Our protocol consists of adding
1/10 vol 3 M NaOAc pH 4.0, vortex, then 1 vol phenol, vortex, then 1/10 vol
chloroform. It is upon adding the chloroform when we usually get a phase
separation, but not in the HeLa lysates. In order to obtain a phase it was
necessary to add chloroform in the same amount as the phenol.
The number of cells being used for lysis in suspension cultures is 4 X 108
cells in 5 ml of guanidium lysis buffer. The lysate clears up fine, so we feel
the amount of buffer is sufficient. The ratio of lysis buffer to cell number
is consistent with what we use for monolayer cultures.
Any ideas on why we're losing our 28S? We're stumped!
Thanks.
The Weber Lab
