RNA extraction from larvae
r.dewhurst at rhbnc.ac.uk
Tue Aug 13 10:13:51 EST 2002
I am having problems getting good quality RNA from zebrafish
larvae (10 dph). I was wondering if anyone could help me trouble-
shoot and give me an idea of a 'good' yield.
I am using a rotary homogeniser to powder approx 50 frozen larvae
in a microfuge tube. The sample is then lysed in a guanidinium
solution. At this stage the black pigmented material forms a smear
on the side of the tube. I spin down at 10,000 rpm 2 mins 4 degC
and remove as much debris as possible (mainly the black
material). I then sequentially extract with Phenol:Chloroform:IAA
and Acid-Phenol:Chloroform. Precipitation is with isopropanol and
resuspension in DEPC treated water.
>From fifty larvae I am only recovering 10 ug of total RNA.
Spectrophotometer ratios indicate that the sample is contaminated
(260/280 nm 3.87 and 260/230 nm 3.13). An aliquot run on an
agrarose gel indicates that the RNA is not degraded..
I would appreciate any advice on how to improve the protocol or
alternatives. I would also be interested in how much RNA I should
be able to extract from a known number of larvae.
Thank you for your time,
School of Biological Sciences
University of London
Tel +44 1784 41 41 96
Fax +44 1784 47 07 56
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