RNA purification

Tina anfortin at webnet.qc.ca
Thu Aug 3 20:29:20 EST 2000


I have already done RNA purification with the TRIZOL reagent on 80X106 of
PMN and the yield was good (for this type of cell e.g. approx. 25 ug) and
the ratio too. Now I want to isolate RNA on mini-prep scale e.g. 10X106
cells per tube (ependorff 1.5 mL) because I will have many different
conditions to do in the same experiment so in this way it will technically
more convenient to do.
However, scaling this easy technique from a large-prep to a mini-prep seems
not to be evident. Yield is OK for 10 millions of cells but ratio 260/280 is
very bad (e.g. 1.0) ! I  followed the proportion of cells versus Trizol
suggested by the company e.g. 1 mL Trizol per 10 millions of cells. Whats
wrong??
Do I have to put 1.5 mL of Trizol for 10 millions of cells or isolate RNA
with a commercial kit?
I try to get a ratio around 1.6-1.7 because many people said to me that for
doing semi-quantitative RT-PCR, I need high quality RNA (high quality being
not necessary required for a technique such as Northern Blot).
Thanks any comments again !!!
Excuse my numerous questions, but I'm a beginner in this field.
Tina







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