Introduction

Isolation of intact RNA from the pancreas has always represented a particular challenge for molecular biologists interested in gene expression in this tissue. This is clue to extremely high levels of RNase A, estimated for rat pancreas at 200 µg/g tissue (1), that are present in secretory granules disrupted during the isolation procedure. The technique of Chomczynski and Saachi (2), which involves lysis of cells in the strongly denaturing cocktail of guanidine isothiocyanate and phenol, has proven to be a highly efficient method of preparing intact RNA from the majority of tissues and is still the method of choice for most applications. However, it gives, at best, variable results with the pancreas, even when combined with the use of RNase inhibitors and cold-temperature homogenization (3,4) or immediate snap-freezing of the pancreas (5) prior to grinding the frozen tissue in a denaturing solution like TRIZOL® reagent.

The major problem with all these techniques is that RNA degradation appears to commence during dissection of the animal and removal of the pancreas. Therefore the major variable is likely to be the efficiency with which the tissue is removed and exposed to RNase inhibitors. To address this problem, we have developed a novel method for RNA isolation that involves pretreatment of the mouse pancreas by in situ ductal perfusion with RNAlater® prior to removal of the tissue. The RNA was subsequently isolated using TRIZOL reagent and was obtained in consistently high yield. A comparison of pancreatic RNA isolated by this method to RNA isolated from NIT-1 pancreatic β cells using the TRIZOL method confirmed that RNA of equally high quality was isolated by this novel approach. When coupled with the Chomczynski and Saachi method (2), this perfusion step significantly improves the reliability of isolation of high-quality pancreatic RNA.

Materials and Methods

Animal Care and In Situ Ductal Perfusion of Mouse Pancreas with RNAlater

SJL/J mice (5- to 6-week-old), weighing 18–20 g (from Jackson Laboratories, Bar Harbor, ME, USA) were housed in a certified animal care facility and handled according to the Guide for the Care and Use of Laboratory Animals (6).

All equipment used in organ dissection was first treated with RNaseZap® (Ambion, Austin, TX, USA) according to the manufacturer's instructions. To circumvent RNA lysis by RNases that may be released in the mouse pancreas when the animal is stressed, all procedures were conducted as swiftly as possible after each mouse was sacrificed. The mice were anesthetized with Avertin® consisting of 1.25% (v/v) 2,2,2-tribromoethanol, 2.5% (v/v) 2-methyl-2-butanol in distilled water at a dose of 0.02 mL/g body weight administered intraperitoneally (7). the mice were then euthanized by cervical dislocation. The abdominal and chest cavities were opened, and the lower lobes of the liver were moved aside to expose the pancreas and bile duct. Under a Model SMZ645 stereoscopic zoom dissecting microscope (4× magnification; Nikon Canada, Mississauga, ON, Canada), the bile duct was clamped with a bulldog clamp at the Sphincter of Oddi, where it enters the duodenum. A 3-mL syringe with a slightly bent 27-G needle was inserted into the bile duct near the liver and 1 2 mL RNAlater (Ambion) was slowly injected ((Figure 1)). As the pancreas was perfused, it visibly ballooned and became more translucent. Once completely perfused, there was resistance to injecting further liquid. At tiffs time, it was excised with scissors and placed in 5 volumes of RNAlater on ice. The time to perfuse the pancreas was found to be <1 min in the hands of an experienced technician. The technique is similar to collagenase perfusion for islet isolation. In cases where snap-freezing of the pancreas was performed, the tissue was placed in a precooled 1.5-ml cryovial, and this was immediately put in liquid nitrogen.

Figure 1.

Isolation of Total RNA

Extraction of total RNA from 5 × 10⁶ NIT-1 pancreatic β cells (ATCC, Manassas, VA, USA) was conducted in parallel to RNA isolation from the perfused pancreas. Previous experience had demonstrated that we could obtain high yields of RNA from NIT-1 β cells using TRIZOL (Invitrogen, Carlsbad, CA, USA) (8), and these cells were therefore used as our positive control for RNA yield and integrity. NIT- 1 cells were cultured in F12/K medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 1% penicillin, streptomycin, and neomycin. The cells were harvested in 1 mL TRIZOL prior to RNA isolation. The perfused pancreas was placed in 5 mL TRIZOL and homogenized for 20 s using an Ultra Turrax® T18 basic homogenizer (IKA Works, Wilimington, NC, USA) at level 4. RNA was then isolated from an aliquot of the homogenized pancreas and the NIT-1 cell pellet, as described below.