Abstract 819: Ambient temperature storage of RNA for use in a RT-qPCR test for the detection of GCC mRNA

Abstract

Abstract The purpose of this study was to evaluate the integrity of T84 cell and lymph node RNA samples, and their use in an RT-qPCR-based diagnostic test, following storage in a chemical matrix designed to stabilize RNA and slow the action of RNase at ambient temperature. Total RNA was purified from the T84 human colonic adenocarcinoma cell line (T84 cells), and from formalin-fixed paraffin-embedded (FFPE) lymph node (LN) tissue. 5µg aliquots of RNA were applied to GenTegra™ RNA tubes, which contain an inert chemical matrix designed to stabilize and protect RNA. RNA samples were stored in the presence or absence of the chemical matrix for 24 hours in the liquid state at 25°C and 37°C, and for two weeks in the dry state at 25°C, 37°C and 56°C. A set of RNA samples was stored at −70°C as a control. Following storage, recovery yields and RNA integrity numbers (RIN) were compared with frozen controls. Transcript-specific cDNA synthesis was performed and the cDNA products were used to conduct qPCR using primers and TaqMan probes targeting β-Actin (ACTB) mRNA for the T84 specimens or using primers and Scorpions probes targeting GCC and ACTB mRNA for the lymph node specimens. T84 cell RNA was incubated in the liquid state at 25°C or 37°C for 24 hours in the presence and absence of a chemical matrix and compared to a control samples stored at −70°C. There was no difference in the RIN between the samples stored at 25°C or 37°C in the presence of the chemical matrix and the frozen control. A decrease in RIN was observed for samples stored at 37°C in the absence of chemical matrix. T84 cell and LN RNA was dried overnight in the presence or absence of a chemical matrix and stored at 25°C, 37°C or 56°C for comparison with a control stored at −70°C. Quantitative recovery was achieved following rehydration of the dried RNA. The RIN of samples stored in the presence of the chemical matrix for two weeks at up to 56°C were not significantly different from control RNA stored at −70°C. Obvious degradation was observed in RNA stored at 25°C, 37°C or 56°C in the absence of chemical matrix. RT-qPCR analysis of the ACTB transcript revealed no difference in Ct values for T84 cell or LN RNA samples stored dry in the presence or absence of chemical matrix and controls stored at −70°. When LN RNA was used for a RT-qPCR diagnostic test for detection of the GCC transcript, the Ct values and copy numbers did not differ significantly between samples stored dry in the presence or absence of a chemical matrix and controls stored at −70°. In this study, we demonstrate that an inert chemical matrix designed to protect against oxidation and hydrolysis and to slow the action of RNase, stabilized RNA samples in the liquid state at 25°C and 37°C for 24 hours, and in the dry state at 25°C, 37°C and 56°C for two weeks. RNA integrity equivalent to frozen controls was maintained during the storage periods, and the chemical matrix had no effect on the results of an RT-qPCR based diagnostic test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 819.