Introduction

The value of RNA as a tissue analyte has been a persistent technical concern, due to its short half-life in vivo and in vitro. RNA instability results from the profound susceptibility of RNA to hydrolysis. RNA also becomes vulnerable to oxidation under more extreme conditions, such as long-term storage or high temperature. The development and validation of high-value RT-qPCR–based cancer molecular tests requires extraction of RNA from many tissue specimens that are harvested during surgery. Thus, there is a need for a cost-effective, easily accessible storage system for purified RNA. However, frozen storage is a costly and inefficient method of preserving the increasing number of purified RNA specimens. In addition to storage costs, diagnostic laboratories also face sample handling issues when working with irreplaceable patient RNA samples. Even in the best RNA preparations, degradation often begins the moment the RNA is purified from a tissue sample, requiring the RNA to be handled on ice in an effort to slow the rate of degradation.

A simple solution for RNA stabilization

A new product for the stabilization of purified RNA was recently introduced by GenVault Corporation. GenTegra™ is an inert chemical matrix that inactivates trace RNase and protects RNA samples from hydrolysis and oxidation, enabling long-term storage and transport of purified RNA in the dry state at ambient temperature, and providing an added level of stability for RNA in the liquid state during sample handling. GenTegra™ is provided in convenient, ready-to-use aliquots in 0.3-mL cluster tube or 1.5-mL screw cap tube formats (Figure 1).

Figure 1. GenTegra™ screw cap tubes (left) and 0.3ml cluster tubes (right) contain an inert chemical matrix that stabilizes purified RNA at ambient temperature. (Click to enlarge)

GenTegra™ is compatible with RNA purified from sources including blood (e.g., PAXgene) and tissue using standard kits. RNA recovered from GenTegra™ has been analyzed using the most demanding molecular assays, including expression profiling on leading platforms, and it has been shown to have no effect on RT-qPCR analysis. The quality of RNA assessed via gel electrophoresis and Agilent Bioanalyzer analysis is equivalent to samples stored frozen. Quantitative (99.9%) recovery of dried RNA is achieved by simply adding water.

Storage of RNA in GenTegra™ eliminates damage from repeated freeze-thaw cycles and reduces the need for frozen storage of RNA, enabling organizations to achieve their sustainability goals by greatly reducing the number of kWh/year used for cryostorage. With GenTegra™, it is possible to transport RNA in the dry state at ambient temperature. GenTegra™ imparts thermal stability to RNA, preserving samples at temperatures ranging from −80°C to 76°C, which may occur during shipping. Significant savings in time and money can be achieved simply by eliminating the need for shipping on dry ice. The stability of purified RNA is increased by a unique formulation of chemical RNase inhibitors that enables handling at room temperature even in the presence of trace RNase. Here, we evaluated the recovery yield, quality and integrity of purified RNA samples following storage in GenTegra™ tubes, and their performance in a RT-qPCR-based cancer molecular test.

Materials and methods

Total RNA was purified from the T84 human colonic adenocarcinoma cell line (T84 cells). Human lymph node (LN) RNA was purified from formalin-fixed paraffin-embedded (FFPE) tissue specimens, as described (1). An aliquot of each RNA sample was stored at −70°C as a frozen control. Five micrograms of RNA were applied to GenTegra™ tubes, dried overnight, and stored in the dry state for 2 weeks at 25°C, 37°C, or 56°C. Samples were rehydrated in 20 μL water. RNA was quantified using a NanoDrop 1000. Complete electropherogram and RNA Integrity Number (RIN) were obtained using the RNA 6000 Nano Chip on the Agilent 2100 Bioanalyzer. Transcript-specific cDNA synthesis was performed in 20-μL reaction volumes with 1.25 μg RNA, and 5-μL aliquots of cDNA products were used in triplicate to conduct qPCR as described (1).

Results

RNA aliquots were dried overnight in the presence or absence of GenTegra™ and stored at 25°C, 37°C, or 56°C for comparison with a frozen control stored at −70°C. Storage at elevated temperature was used to assess the ability of RNA samples stabilized in the GenTegra™ matrix to withstand temperatures of up to 60°C, which can occur during shipping (2). Following rehydration, there was no significant difference in percent recovery between samples stored in the presence of GenTegra™ and frozen controls. The RINs of samples stored with GenTegra™ for 2 weeks at 25°C or 37°C were not significantly different from control RNA stored at −70°C (Table 1). Even storage at 56°C did not significantly alter the RIN when GenTegra™ was present, with a difference of only 0.8 between samples stored at 56°C with GenTegra™ and frozen controls. Conversely, significant degradation was observed in RNA stored in the absence of GenTegra™. T84 cell and LN RNA samples were then subjected to RT-qPCR analysis of the β-actin (ACTB) transcript. There was no significant difference between the ACTB Ct values for samples stored dry in the presence or absence of GenTegra™ matrix and frozen controls. LN RNA samples were further analyzed via a RT-qPCR–based cancer molecular test. As with ACTB, the Ct values and copy numbers did not differ significantly between samples stored dry in the presence of GenTegra™ matrix and frozen controls. The complete results can be viewed at www.genvault.com/RNA.pdf.

Table 1. RIN results of T84 cell RNA stored in the presence or absence of GenTegra™ in the dry state for two weeks at 25°C, 37°C, and 56°C, and a control stored at −70°C. (Click to enlarge)

Conclusions

In the present study, we examined the use of GenTegra™ for storage of total RNA purified from the T84 human colonic adenocarcinoma cell line and from FFPE human lymph nodes. Here, we have shown that the integrity of RNA stored in the dry state at ambient temperature is preserved in the presence of GenTegra™. The substantial degradation observed in samples stored without GenTegra™ at 37°C and 56°C demonstrate the ability of GenTegra™ to protect against heat-catalyzed hydro-lysis and oxidation of the RNA. GenTegra™ eliminates concerns about sample loss due to high temperature or shipping delays. Two types of RNA were stored with GenTegra™; high-integrity T84 cell RNA (RIN above 9.00) and RNA recovered from FFPE lymph nodes, which was highly degraded (RIN of 2.30). Both types of RNA were stabilized during a 2-week storage period at 25°C, 37°C, and 56°C in the presence of GenTegra™, indicating that GenTegra™ has the ability to stabilize RNA samples of various quality and purity. Both T84 cell RNA and lymph node RNA exhibited similar performance to the frozen control in RT-qPCR for ACTB. Moreover, results obtained from an RT-qPCR–based molecular test were identical for samples stored in the presence of GenTegra™ and frozen controls. Thus, GenTegra™ does not inhibit RT-qPCR. In conclusion, GenTegra™ enables storage and transport of purified RNA at ambient temperature and is compatible with RT-qPCR– based molecular tests. Thus, GenTegra™ assures preservation of irreplaceable RNA samples without interfering with downstream analyses.