Fish tissue RNA purification

In general the use of guadinium/phenol based kits such as Triazol for Sigma give better results than this, the original method. But it can be expensive for large numbers of samples. So for what its worth, here is my original methods with a slant toward fish RNA sources.
 


Notes

RNA homogenisation buffer is 4.5M guanidinium thiocyanate (or 6M guanidinium hydrochloride), 25mM sodium citrate, pH 7.0, 0.5% sarkosyl stored at room temp with DTT added to 10mM from frozen 1M stock immediately before use.

Alternatively use a commercial mix (eg Triazol or RNAzol) which are essentially the same as described above but can give better results ( for a lot more cost)

It is important to wear gloves at all stages, firstly to afford protection from guanidinium thiocyanate and phenol and secondly to minimise RNAase contamination.

MilliQ (or eqivalent grade) water is OK to use directly if collected in factory sterile plastic. DEPC can be used to treat solutions and plastics or glass if required. DEPC treatment of solutions involves adding 0.1% DEPC to the solution, shaking for 1 hour and autoclaving. Tips and vials are shaken in suitable containers in a suitable volume of 0.1% DEPC/ water for 1 hour, drained and autoclaved. Excess moisure may be removed by leaving in the drying cabinet with  loose lids for a while.

Repeated washes with 70% ethanol can often result in cleaner preps.

Attempting to homogenise too much tissue in too small volumes of buffer will result in poor preps, RNA pellets resistant to resuspension, and low purity ratios. A 10% (w/v) homogenate is about the upper limit for good liver and larval RNA.

Drying RNA pellets too much will also make them resistant to resuspension although traces of ethanol can give similar problems. Therefore it is recommended that pellets are allowed to dry in air for about 15 mins or longer if necessary. Dry means no ethanol left- pellets will still be moist with residual water.

It is critical that all samples are treated equally and great care is taken to avoid RNAase contamination, therefore it is recommended to avoid working up large numbers of samples at one time. It is better to get good samples over a few days than many poor samples in a morning. It is also important to test a previously unused source material to check that it is possible to get good RNA before embarking on a full scale experiment.