by Sharron Ohgi
RNA research has become prevalent due to its role as a gene silencer and in gene expression studies. However, RNA samples are notoriously difficult to work with given their highly labile nature and tendency to degrade even under carefully controlled RNase-free conditions and maintenance in cold environments. Exposure to slightly elevated temperatures for even short time periods can compromise RNA integrity and detrimentally affect downstream assays and results.
Additionally, the necessity to store and transport RNA samples under cold conditions can be very expensive, expending not only significant amounts of laboratory budgets, but energy, too. Transporting frozen RNA samples on dry ice can cost hundreds of dollars.
Direct preservation, stabilization
 Figure 1 |
RNAstable (Biomatrica, San Diego, CA) directly preserves and stabilizes purified RNA samples at room temperature for at least six months and has demonstrated an equivalent of three and a half years stability under accelerated aging conditions.
The product was developed based on the natural principles of anhydrobiosis (life without water), a biological mechanism employed by some multicellular organisms that enables their survival while dry for up to 120 years. Anhydrobiotic organisms such as tardigrades and brine shrimp can protect their DNA, RNA, proteins, membranes, and cellular systems for survival and can be revived by simple rehydration.
The molecular principles of anhydrobiosis have been transferred to a synthetic chemistry-based stabilization technology. RNAstable works by forming a thermo-stable barrier, essentially "shrink-wrapping" and stabilizing the fragile RNA molecule at ambient temperature and providing protection against degradation (Figure 1). It is supplied as a dried matrix on the bottom of 1.5 ml microcentrifuge tubes or in the wells of a microplate. Each sample-storage tube or well contains enough matrix to protect up to 100 µg of RNA. The steps involved in using the product for RNA storage are outlined in Figure 2.
By simply pipetting RNA samples into the tube or well, the dried RNAstable matrix is rehydrated and mixes with the RNA. Through its natural affinity to RNA, the matrix associates with the nucleic acid in the liquid phase. Upon air-drying, the stabilizing barrier that protects the RNA from degradation is formed. Samples can then be stored at room temperature in relative humidity conditions of 50% or maintained in a moisture-barrier container.
 Figure 2 |
Sample recovery simply requires rehydration. Since the rehydration volume can range from 10 to 100 µl, storage of RNA in RNAstable provides a method for sample concentration, eliminating the need for salt precipitations and sample loss due to multiple wash steps.
RNA samples recovered following storage in RNAstable can be used directly in downstream applications such as quantitative RT-PCR, bioanalyzer, microarray analysis, end-point PCR , gel electrophoresis, cDNA synthesis and reverse transcription without inhibition or interference.
To validate the stabilization of RNA stored dry in RNAstable, total RNA was isolated from human 293T cells grown to 90% confluence in T-175 flasks in DMEM supplemented with 10% fetal calf serum at 37 C and 5% CO
2. Cells were dissociated from the flask by incubating with 0.25% Trypsin-EDTA at 37 C for 5 minutes. The cell pellet was stored frozen at -20 C until ready for use. Frozen 293T cells were resuspended in 1 ml of PBS, and total RNA was isolated using the TRIzol isolation protocol (Invitrogen Corp., Carlsbad, CA) following manufacturer's instructions. Isolated total RNA was resuspended in DEPC-treated water and stored at -20 C.
Aliquots of 50 µg of total RNA were applied to RNAstable and allowed to dry for 1.5 hours in a SpeedVac (Thermo Fisher Scientific, Waltham, MA) without heat. An unprotected control sample was prepared by drying 100 µg of total RNA into an empty tube under identical conditions. Samples were then stored for 14 days at room temperature or six months at 50 C to assess long-term stabilization. RNA was rehydrated by adding DEPC-treated water to a final concentration of 1 µg/µl for each sample.
Aliquots of RNA stored at room temperature in RNAstable and control samples stored at -80 C were analyzed with a bioanalyzer. Results indicate that RNA is stabilized and protected in RNAstable, as there is no apparent degradation when compared to the frozen control sample (Figure 3, left panel).
 Figure 3 |
RNA is also protected at elevated temperatures, even after six months at 50 C. Sample storage at 50 C for six months is equivalent to three and a half years at room temperature. The integrity of samples preserved using RNAstable are comparable to freezer-stored control samples, while the unprotected sample was completely degraded (Figure 3, right panel).
RNAstable provides scientists with a high degree of confidence when working with RNA samples, as well as a reduction of the cost associated with storing and transporting RNA using traditional methods. It allows for long-term stabilization of RNA samples at ambient temperatures. Sample recovery is achieved through one-step rehydration, resulting in samples that are ready for immediate use without the need for further purification. Samples can be stored with minimal effort on the bench top and can be shipped at room temperature, greatly reducing reliance on costly freezer units and eliminating the need for dry ice during transport. The integrity of RNA stabilized in RNAstable is protected against fluctuating and inconsistent temperatures commonly experienced during shipment. Sample stability is secured even at elevated temperatures of 50 C.
Author Sharron Ohgi is a research associate with Biomatrica. For more information, contact Biomatrica, 866-379-6879, www.biomatrica.com.