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07/15/09

Welcome to the new version of the real time PCR website. Little information has changed, but the primer list has been updated!

 

Animal RNA isolation

Introduction:  Compared with other RNA preparation techniques, I find that Qiagen's RNeasy kit provides superior RNA.  An alternate (less expensive) method is TriReagent, sold by Sigma.  Tissues can be stored in RNAlater (Promega) until you are ready to isolate them, but it is important to make small pieces so that the RNA later can permeate the tissue and prevent RNase action.

1.  Before beginning, prepare tubes containing 700uL of RLT buffer, prepared as described in the RNeasy protocol book (don't forget the beta-mercaptoethanol).

2.  Prepare a polytron with DEPC-treated water, which prevents RNA degradation.   

3.  As the tissues are harvested, cut a small piece off, roughly the size of a grain of rice.  Immediately place in the RLT buffer and grind for 30 seconds. 

4.  Transfer the tube to ice until you are finished.  Samples can be stored in the -70C for a about a month.  (Note, Qiagen doesn't recommend this, but I've found that tissues do not degrade in RLT buffer over this short period of time.)

5. Prepare RNA samples as described in the Qiagen RNeasy kit.