Determination of RNA stability using reverse transcription real-time PCR

Abstract

The aim of this study was to verify an effect of RNA storage in different laboratory conditions. Especially, this work was focused on the importance of using diethylpyrocarbonate (DEPC) treated water for storage of isolated RNA. The effect of storage of RNA samples in different temperatures was monitored according to various times as well. Isolated RNA was incubated at 20 °C, 4 °C, −20 °C and −80 °C, whereas the temperature −80 °C was used as a control. After incubation only mRNA was converted to cDNA by reverse transcription. The polymerase chain reaction in real time (real-time PCR) was used for a measurement of RNA degradation. No statistically significant interactions were found between RNA treatment conditions if analysis of variance (ANOVA) model was applied. The result showed that storage of isolated RNA in water treated with DEPC is not necessary. This approach prevents possible inhibition downstream reaction caused by DEPC. The results of this study can be used in all molecular applications based on RNA.

Keywords

• RNA
• RNases
• diethylpyrocarbonate
• real-time RT-PCR