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RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue
Published on: August 2, 2014
RNA isolation from mouse pancreas: a ribonuclease-rich tissue
Ana Clara P Azevedo-Pouly1, Ola A Elgamal1, Thomas D Schmittgen2
1Pharmaceutics & Pharmaceutical Chemistry, The Ohio State University.
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.
Insights
Isolating high-quality RNA from mouse pancreas, a ribonuclease-rich tissue, requires specific protocol modifications. These key steps ensure RNA integrity suitable for gene expression analysis.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Mouse pancreas is rich in ribonuclease (RNase), posing challenges for high-quality RNA isolation.
- High RNase levels can degrade RNA, hindering downstream molecular analyses.
Purpose of the Study:
- To develop and optimize a protocol for isolating high-quality RNA from mouse pancreas.
- To overcome the challenges posed by high endogenous RNase activity.
Main Methods:
- Modified standard phenol/guanidine thiocyanate lysis reagent protocols.
- Employed a high lysis reagent to tissue ratio.
- Included removal of undigested tissue and addition of a ribonuclease inhibitor.
Main Results:
- Successfully isolated RNA from mouse pancreas with an RNA Integrity Number (RIN) greater than 7.
- Achieved RNA suitable for routine gene expression analysis.
- Demonstrated the protocol's effectiveness against high RNase levels.
Conclusions:
- Modified protocols are critical for isolating RNA from ribonuclease-rich tissues like the mouse pancreas.
- The developed method yields high-quality RNA suitable for gene expression studies.
- This protocol can likely be adapted for other RNase-rich tissues.
