I am interested in extracting RNA from eyes, liver, intestine and gills of adult zebrafish. Following TriZol extraction, I run 500ng on a gel and sometimes I get nice strong bands whereas at other times I can see the bands only if I turn up the intensity of the UV. Even for the same type of tissue I get a lot of variability.
Upon dissection, I immediately place the tissue in TriZol and then homogenise it with an electric homogeniser. If I have to store it, then I store the TriZol with the tissue at -80. Other than this, I am following the protocol as described.
Any suggestions? Thank you
One thing you could try is to increase the amount of TRIzol you use, especially if you have a bit more tissue in some cases. More TRIzol will facilitate lysis of the tissues and ensure proper phase separation, which will prevent carry-over of RNases and other proteins into the aqueous phase of the RNA preparation.
Any RNA purification from tissue is more difficult. Consistency in tissue size and optimization of amount of TRIzol (mentioned above) and homogenizing are critical factors. Also, I've found considerably higher RNA yields from Trizol preps if you incubate at -20 for at least 20 min after adding the isopropanol.
In this case you may try to do RNA re-precipitation.. butt when you do it with one sample.. do it with all even with those which give good bands. so that you are sure that you have treated all the samples in the same way.
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