RNA extraction from larvae

[Rachel Dewhurst]

Dear All,

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,

James

James Wheeler
School of Biological Sciences
Royal Holloway
University of London
Egham
Surrey
TW20 0EX
UK

Tel  +44 1784 41 41 96
Fax +44 1784 47 07 56