A week back I had enquired about RNA extraction and here is a copy of that post:
I am looking for some feedback on the use of conventional RNA extraction protocols versus the use of the commercially available columns (eg. Pharmacia, Qiagen etc.) when you are dealing with precious little samples. One of our grad students haven't had much luck with the Qia column the one time she tried. I will be shortly extracting some RNA from small amounts of *plant* tissues. Any pointer, experiences, tips etc. will be greatly appreciated. The RNA will be further enriched for polyA+ for, perhaps, cDNA construction..."
Few responded to it and here is a summary of those responses:
We have just published a method in Biotechniques (January issue-Eaton et
al) that gives RNA signal from a single cell.
It utilises Nonidet P-4O lysis in the presence of RNAsin and DTT.
It does not eliminate DNA but all RT-PCR primers shoud be designed so that
the PCR product is absent (or at least distinct from the expected product)
when DNA is used as a control.
We use Superscript II in the paper but MMLV also works just fine and is a
In your case if you pulverise the frozen tissue you may be able to leave
out the Nonidet P-40 and just add the RT-PCR mix making sure that RNAsin
and DTT are present to inhibit the RNAses.
Try SIGMA's TriReagent. Phenol based, isolates total RNA with very good
Try Novagen's magnetic beads.
In handeling precious little sample of RNA, I do not know of any truely reliable
methods that are not commecial. However, in talking with a scientist yesterday
from Genetech, I learned that Behringer-Mannheim has a product that consists of
a strepavadin-biotin poly dT bound to the inside surface of PCR tube. A cell
lysate is added and incubated. They lysate is then removed, the tube washed,
and the RT coctail added to generate cDNA. The sample is then further amplified
by PCR. My suggestion is to contact BMs technical support group and inquire.
I would be interested to learn the outcome of your inquiry.
Thank you for all the responses. I will try to follow up on further queries.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu