The first part of this article summarizes findings from three peer reviewed journal articles, each of which cites the use of Ambion's RNAlater and describes how RNAlater can be a real advantage when tissue is difficult to collect or work with. Samples derived from nasal swabs, postmortem tissue, and finely dissected blood vessels are all described. In the second citation, researchers were able to look at protein levels as well as RNA in the tissue. In the third article, the change in sample texture -- in this case stiffening of the blood vessels -- made fine dissection much easier.
In the second part of this article we present another example of how RNAlater can facilitate the tissue collection process when it is complicated by the need for fine dissection or tissue punching. By stabilizing the RNA first, there is no longer any pressu re to proceed quickly through a difficult collection process. RNAlater not only makes tissue collection easier, but it can actually enable the collection of tissues for RNA isolation that could not previously be subjected to RNA analysis.
Make Tissue Collection Easier by Using the Tissue Storage Solution, RNAlater
Dreskin SC, Dale SN, Foster SM, Martin D, Buchmeier A, Nelson HS (2002) Measurement of changes in mRNA for IL-5 in noninvasive scrapings of nasal epithelium taken from patients undergoing nasal allergen challenge. J Immunol Methods 268: 189-95.
Scrapings of the nasal epithelium provide a simple, non-invasive way of collecting samples to measure changes in gene expression following allergen challenge. In a study by Dreskin and colleagues, 10 patients were challenged with velvet grass or ragweed extracts, after which 2 scrapings of the nasal epithelium were collected. The collection sticks used for the scrapings were immediately immersed in RNAlater and stored at 4C. Insufficient RNA was obtained from the scrapings of two of the patients. However the remaining patients' samples yielded approximately 150 ng/scraping. The RNA, which resolved into "tight" 18S and 28S ribosomal RNA bands upon gel electrophoresis, was used to analyze IL-5 mRNA levels by RT-PCR. IL-5 mRNA levels were found to correlate with patients' scores of late-phase allergic symptoms, emphasizing the value of RNA analysis for such studies.
De Paepe ME, Mao Q, Huang C, Zhu D, Jackson CL, Hanson K (2002) Postmortem RNA and protein stability in perinatal human lungs. Diagn Mol Pathol 11(3): 170-6.
Previously, use of postmortem fetal and neonatal human lung tissue for RNA analysis has generally been thought to be impossible due to RNA degradation in postmortem tissues. Published results to the contrary with brain tissue led De Paepe and colleagues to examine the quantity and quality of RNA obtained from postmortem fetal and neonatal human lung tissue stored in RNAlater upon collection or snap-frozen in liquid nitrogen at autopsy. They found no difference in the quality of the RNA obtained via the two storage methods as determined by A260/A280 ratio, gel electrophoresis, and RT-PCR analysis of gene expression (-actin and SP-B). While the RNA quality was comparable, the RNA yield was found to be significantly higher in tissues that were stored in RNAlater vs. those that were frozen. Similar results (same quality, better yield) were obtained for protein isolated from tissues stored in RNAlater compared to those frozen in liquid nitrogen. In addition to obtaining high quality/quantity RNA, the authors pointed out the practical advantages of tissue storage in RNAlater, namely that tissues in RNAlater can be stored at room temperature, allowing for easy integration of this collection procedure into complex autopsy protocols. The tissue is also easily transported, thereby facilitating interinstitutional collaborations.
Fine Dissection of Tissue Samples for RNA
Rodrigo MC, Martin DS, Redetzke RA, Eyster KM (2002) A method for the extraction of high-quality RNA and protein from single small samples of arteries and veins preserved in RNAlater. J Pharmacol Toxicol Methods 47: 87-92.
The veins and arteries comprising the mesenteric vasculature play a critical role in cardiovascular homeostasis. It is therefore of interest to examine gene expression in these vessels for various cardiovascular studies. For such studies it is important to separate veins from arteries (and to remove contaminating adipose, nerve and connective tissue) since they function differently under normal conditions and respond differently to drugs. The time required for such intricate dissection and the high levels of RNases in these tissues generally results in RNA degradation. Fortunately, Rodrigo et al. report the prevention of RNA degradation in rat mesenteric vasculature following dissection and storage of the mesenteric arcade in RNAlater. Coarsely dissected tissue was stored at -20C in RNAlater for a minimum of 3 days. Storage resulted in a stiffening of the vessels that further eased dissection. Tissues were then submerged in RNAlater and spread out in a Petri dish placed on ice. Finely dissected tissues were placed in fresh RNAlater and stored at -80C until RNA isolation. No usable RNA was obtained in the absence of RNAlater - "at best, a blur of degraded RNA could be seen on an ethidium bromide-stained gel". With RNAlater, however, high quality RNA was obtained as determined by A260/A280 ratio (1.8-2.2) and gel electrophoresis.
RNAlater Enables Sampling of Brain Regions
RNAlater Tissue Collection:RNA Stabilization Solution has become indispensable for preserving tissue and cell samples prior to RNA extraction -- particularly when samples are collected where there is no access to freezers or liquid nitrogen. Dr. Ambrose Dunn-Meynell, working in the lab of Dr. Barry E. Levin at the VA Medical Center in East Orange, New Jersey has found another application for RNAlater, using it to facilitate fine tissue dissection of rare brain tissue.
To analyze the gene expression pattern in different regions of rat brain, Dr. Dunn-Meynell collected tissue punches of the arcuate nucleus of the hypothalamus and the ventromedial thalamus. Punches were derived from rat brain tissue that was frozen and cryosectioned to obtain 300 mm sections of the area of interest. The sections were immediately plac ed in RNAlater and incubated at 4C overnight. Subsequently, regions of interest were punched from the section using a blunt-end hypodermic needle. Ambion's RNaseZap was used to clean the needle and eliminate RNase contamination. Both fresh and frozen brain tissues are normally very flimsy and sticky, which cause difficulty in obtaining intact tissue punches. However, RNAlater makes brain tissues more durable, thus more ammenable to needle dissection.
After purification of total RNA from each brain punch using Ambion's RNAqueous Total RNA Isolation Kit, real-time RT-PCR was used to compare the expression of multiple genes. As shown in Figure 1, the long form of leptin receptor mRNA was detected in arcuate but not the thalamus. Glucokinase, however, was expressed in both arcuate and thalamus at a similar level. For more information about RNAlater, click here.
Figure 1. Comparing Rat Arcuate and Thalamus Gene Expression Patterns. Real time RT-PCR of each of the two samples (arcuate and thalamus RNA) was performed for 40 cycles for both leptin receptor and glucokinase. A lower Ct value indicates a higher expression level for the target gene (it is detected more quickly). P Leptin receptor was not detected (ND) in the thalamus RNA sample after 40 cycles of amplification. This sample was plotted at 40 Ct's for convenience.