RNA extraction for RNAseq - (Aug/21/2015 )
Hello all,
I am trying to extract RNA from embryonic mouse hearts. I have a few questions, what are people's methods of disruption/homogenization-we don't have any tools for that right now, I am wondering if there is anything cheap I can buy. Also, while I do the dissections, is it possible to put the tissue immediately in the RLT that comes with the Qiagen kit? I tried putting it in RNA later, but when I tried to homogenize with a tube mortar and pestle, I couldn't grind the tissue because it got so hard in the RNA later solution. And finally, do people find that the trizol method is better than the RNA kits from qiagen, and if so does anyone have a protocol?
Tara
you can freeze the heart in liquid nitrogen (before or after treating with rnalater) and then pulverize it with either a mortar and pestle or a pulverizer specifically designed for this.
I had used IKA polytron and mortar and pestle for disruption and homogenization of a range of tissues for RNA extraction, both results were equally good.
you can put the tissue directly into buffer RLT making sure you proceed further for homogenizing, once you homogenize it , you can store it at -80 for further steps of RNA extraction although its better to finish RNA extraction at the same time. To my experience, kits are easy and sure way for the beginners to get RNA from tissues and Qiagen kit surely works.
My favorite way is this:
Liquid Nitrogen Cooled Mortar & Pestle
http://www.braintreesci.com/prodinfo.asp?number=3726001
Some image for how to do it:
Then mix the powder of tissue with Trizol or any solution from any KIT for RNA extraction.