May 08, 2020

Public workspaceStorage and Processing of Tissue for bulk RNA Isolation

DOI
dx.doi.org/10.17504/protocols.io.bf52jq8e
  • 1Stanford University
  • NCIHTAN
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Aaron Horning
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bf52jq8e
Protocol Citation: Aaron Horning 2020. Storage and Processing of Tissue for bulk RNA Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.bf52jq8e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2020
Last Modified: May 08, 2020
Protocol Integer ID: 36762
Storing Tissue for RNA Isolation
Storing Tissue for RNA Isolation
After resection, tissue is immediately flash frozen in 50 ml Falcon tube or 2 ml cryovial tubes by placing tissue into tube then simply dropping filled tube into liquid nitrogen.
The tubes are stored on dry ice or -80C until they can be stored in a liquid nitrogen tank
Bulk RNA Isolation
Bulk RNA Isolation
The large tissue pieces are smashed in a freezing cold mortar and pestle with liquid nitrogen. The desired amount of tissue (30mg - 60mg) is then pulverized and swept into a 1.5 ml centrifuge tube sitting on dry ice to prevent thawing of the tissue.
Note
Its ciritically important for RNA integrity to not let the tissue thaw. Thawing will activate the endogenous RNases within the tisses thus depleting the chemical we wish to assay.

Follow the Quiagen All Prep Kit protocol found here: https://www.qiagen.com/us/resources/resourcedetail?id=bbd50261-3b80-4657-ad58-6a5a97b88821&lang=en. Complete the RNA isolation steps.
Breifly, be sure to lyse and homogenize the tissue with RLT buffer with beta-mercaptoethanol with an 18 gauge needle until the needle runs smoothly. Additionally, if once the lysed tissue in RLT appears to be at all yellow and not clear, dilute the lysed tissue in RLT in more RLT buffer. If the RLT is too dense, it will not run through the columns and you'll loose material.
Elute in water and freeze at -80C.