Ethanol: A simple and effective RNA‐preservation for freshwater insects living in remote habitats

Abstract

AbstractThe often proposed preservation method “snap‐freezing” is rather unpractical for sampling in remote areas. Therefore, the effectiveness, feasibility and affordability of alternative fixatives were investigated. Larvae of Orthocladius sp. (Diptera, Chironomidae) were fixed in RNAlater®, RNAfix (self‐made), acetone and ethanol of two different qualities (methylated and molecular grade). Samples were first kept at room temperature for 24 h, then at −20°C for 1 week, at −80°C for 1 month and at −80°C for 6 months. Additionally, long term storage at −20°C for up to 18 months was tested for methylated ethanol and RNAfix. Subsequently evaluated RNA quality and quantity did not differ significantly between the preservation methods. Methylated ethanol, being the most economical alternative, was chosen to be tested in a field trip. Diamesa latitarsis larvae (Diptera, Chironomidae) and Baetis alpinus nymphs (Ephemeroptera, Baetidae) were collected in two remote streams at 2208 m a.s.l. Samples were cooled (∼ 4°C) for 23 up to 30 h during sampling and transportation. Organisms were identified and photographed in ethanol at room temperature and then stored at −80°C for 6 months until RNA isolation. RNA quality was high and the yield was suitable for down‐stream applications such as cDNA synthesis and real‐time quantitative PCR. Due to these results we recommend methylated ethanol as an inexpensive but reliable RNA‐fixative for sampling in remote areas even if time‐consuming species identification has to be done prior to RNA‐extraction.