RNA isolation from mouse pancreas: a ribonuclease-rich tissue

J Vis Exp. 2014 Aug 2;(90):e51779. doi: 10.3791/51779.

Abstract

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Mice
  • Pancreas / chemistry*
  • Pancreas / enzymology*
  • Pancreas / surgery
  • RNA / isolation & purification*
  • Ribonuclease, Pancreatic / chemistry*
  • Ribonuclease, Pancreatic / metabolism

Substances

  • RNA
  • Ribonuclease, Pancreatic