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

Abstract

The 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.

Methodological Insight Into Mosquito Microbiome Studies

“…The better performance of non-freezing storage conditions may be a result of avoiding freezing-thaw cycles during sample manipulation. The finding of absolute ethanol as an efficient RNA preservative may seem surprising, but it has previously been shown for insect larvae and nymphs (Astrid et al, 2016). In general, our results agree with previous studies where NAP buffer was found as an efficient nucleic acid preservative for various samples (e.g., rat tissues: Camacho-Sanchez et al, 2018;and frog tissues Montero-Mendieta et al, 2017), including those for microbiome studies (e.g., fecal samples: Menke et al, 2017).…”

“…Methodology optimization also involves sample preservation. All methods evaluated here were able to preserve DNA (i.e., needed for microbiome profiling) and RNA (i.e., needed for virus screening), in agreement with previous studies performed with different kinds of samples (e.g., freshwater insects: Astrid et al, 2016; and mammalian blood and tissues: Camacho-Sanchez et al, 2013). Significant differences among treatments were found for RNA yields, but not for DNA.…”

Sensitivity and specificity of real‐time PCR and bacteriological culture for francisellosis in farm‐raised Nile tilapia (Oreochromis niloticus L.)

“…Ethanol has been used as a fixative to preserve nucleic acid for molecular tests in algal (Eckford‐Soper & Daugbjerg ), insects (Astrid, Margit & Leopold ), fish (Borzym, Maj & Matras ) and terrestrial animals (Debroy et al . ).…”

Assessment of RNA extraction protocols from cladocerans

“…Another factor that can influence the quality and integrity of RNA used for downstream analysis besides the extraction method is the preservation of RNA samples. Previous studies have utilised several preservation methods, including preservation in RNAlater and storage at -80°C [ 44 , 51 , 54 , 62 – 67 ], preservation in lysis reagent and storage at -80°C [ 51 , 54 , 62 , 64 ], preservation in ethanol and storage at -80°C [ 38 , 68 ] and immersion in liquid nitrogen and storage at -80°C [ 62 , 63 , 65 ]. Preservation in formalin or 95–100% ethanol and flash freezing by using liquid nitrogen is a conventional method and most regularly used for bulk fixation of zooplankton samples [ 69 , 70 ].…”

“…This assessment can assist scientists in recognising biological pathways and identifying the genes that regulate cell behaviour, development and disease [ 37 ]. However, the consistent and comprehensive analysis of gene expression depends on the quantity, quality and integrity of extracted RNA of the sample [ 38 ]. Subsequently, RNA extraction methods can be divided into two primary categories: conventional phenol-chloroform methods and silica-based kits [ 39 ].…”