I am trying to identify alternative splicing variants of my gene, which is highly expressed in brain. So what kind of brain tissue, frozen, fixed or fresh would be the best for RNA extraction, and later qPCR? And how much of it?
We use a phenol/quanidinisothyiocyanat/chloroform methods for RNA isolation (just the TriFast from peqlab but any similar reagent would work). We are working with mouse brain and directly after preparation of the brain (we perform a fast post mortem perfusion with PBS) we cut it in several parts and directly add the TriFast. We are then roughly homogenising mechanically. This works really fast and easy. Our smallest parts are the bulbus olfactorius or the brain stem. Then we either store them at -80°C for later use or go on with the isolation/precipitation. This work best in our hands: Just to directly lyse the brain in the reagent used for isolation even if we just want to store our samples.
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