RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Summary

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

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.

Introduction

Isolation of RNA with high integrity is required for routine molecular biology experiments such as northern blotting ⁹, qRT-PCR ¹ or gene expression profiling ⁵. Most contemporary methods of RNA isolation are based upon modifications of the guanidine thiocyanate protocols ^{2, 3}. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. The popular method of Chomczynski and Sacchi ³ combined phenol to the guanidine thiocyanate lysis solution, reducing the isolation time to about 4 hr. Many commercially-available RNA extraction reagents are b....

Protocol

1. Preparation

The following practices adhere to the policies set by the Institution’s Animal Care and Use Committee (IACUC).

1. It is recommended to isolate no more than 6 mouse pancreases in any given day. Have all surgical instruments clean and autoclaved, one set per animal.
2. Label all microcentrifuge tubes. Four 2 ml tubes per animal.
3. Place 8 ml of lysis reagent in 50 ml centrifuge tubes on ice. Note: While the purification kit comes with a guanidine thiocyanate phenol reagent that is suitable for pancreas isolation, lysis reagent is used because of the large volumes that are necessary per isolation.

Results

The total RNA yield from 1 ml of lysis homogenate is 20-40 µg. OD 260/280 ratios are typically around 2.0 and the RIN are consistently greater than 7.0. If the RIN is ≤6, the isolation will need to be repeated. Occasionally, a RIN that is higher than 8.0 is achieved.

The two most commonly used methods of euthanizing mice prior to removal of the pancreas are CO₂ asphyxiation or inhalation of isoflurane. Both techniques are followed by cervical dislocation. Since it is possible that t.......

Discussion

Isolating RNA from tissues that are ribonuclease rich represents a great challenge for molecular biology experiments. Various reagents and kits are commercially available that are primarily based upon the phenol guanidine thiocyanate method of RNA extraction. Guanidine thiocyanate denatures protein and thus reduces the activity of ribonuclease. However, the sheer magnitude of ribonuclease in pancreas requires additional modifications to standard protocols of RNA isolation. A protocol is reported here that is based on the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Power Gen 500	Fisher Scientific	14-261-03	Homogenizer
5424R	Eppendorf		Refrigerated microcentifuge
Trizol reagent	Invitrogen	15596018	Lysis reagent
Student Vannas spring scissors	Fisher	91500-09	Use to cut pancreas from attached tissues
Surgical scissors, 6 inch	Fisher	08-951-20	Use to cut scin of mouse
Blunt end forceps	Fisher	1381239	Use to hold pancreas while dissecting
Isoflurane USP	Abbott Labs	4/8/5210	Anesthetic
Rnase out (40 U/µl)	Invitrogen	10777-019	Rnase inhbitor
Rnase Away	Ambion	10328-011	General Rnase inactivator
HPLC grade water	Fisher	W5-4	For washing homogenizer blades
Molecular biology grade water	Hyclone	SH30538.03	Elution of RNA
miRNeasy Mini kit	Qiagen	217004	Purification Kit
2 ml microcentrifuge tubes, certified Rnase Dnase free	USA Scientific Plastics	1620-2700
15 ml centrifuge tubes	Falcon	352099
70% ethanol	Fisher
100% ethanol	Fisher
200 µl pipet tips	Rainin	GPL200F	For cleaning homogenizer blades
Chloroform	Fisher	BP1145-1
Nanodrop	Thermo	ND-1000	Spectrophotometer
Bioanalyzer	Agilent		Capillary electrophoresis analyzer to measue RNA integrity
RNAlater	Ambion		RNA Stabilization Reagent
RNeasy spin columns	Qiagen		Spin columns as a part of the miRNeasy Mini kit
Mice	Chales River	Strain C57BL/6	Their age ranged from 4 to 12 months.
Mice	Jackson Lab	strain 129	Their age ranged from 4 to 12 months.