RNA from tiny amounts of tissue?

Alex Dobrovic adobrovic at medicine.adelaide.edu.au
Thu Feb 6 02:26:26 EST 1997


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
lot cheaper.

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.

Alex

Alexander Dobrovic, Ph. D.
Chief Scientist
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia

Affiliate Senior Lecturer
Department of Medicine
University of Adelaide

Tel 61-8-8222 6884
Fax 61-8-8222 6046 (note new numbers as of August 1996)





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