How to preserve and handle fish liver samples to conserve RNA integrity

Eeva-Riikka Vehniäinen,Jussi V K Kukkonen,Aimo O J Oikari,Marja Keinänen,Maiju Ruusunen,Pekka J Vuorinen

doi:10.1007/s11356-019-05033-0

Abstract

As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at − 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.

Highlights

The studies using transcriptomics are becoming more and more common in ecotoxicology
Even the samples that had been preserved at − 20 °C for 6 months had RNA integrity number (RIN) > 7 and could be considered suitable for RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (Table 1)
(ice/liquid nitrogen for 4 h) had an effect on RIN in samples stored at − 20 °C (ANOVA with repeated measures, p = 0.05) but not in those stored at − 80 °C (t test, p > 0.05)

Summary

Introduction

The studies using transcriptomics are becoming more and more common in ecotoxicology. The RNA integrity can be assessed most reliably with microchip gel electrophoresis, using Agilent BioAnalyzer or Bio-Rad Experion (Imbeaud et al 2005). The BioAnalyzer calculates an RNA integrity number (RIN) for each sample using the electrophoresis results (Schroeder et al 2006). Several RIN cut-offs ranging from 3.95 to 8 have been proposed for transcriptomic analyses (Fleige and Pfaffl 2006; Gallego Romero et al 2014; Huang et al 2013; Ibberson et al 2009; Imbeaud et al 2005; Weis et al 2007)

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