Single-animal, single-tube RNA extraction for comparison of relative transcript levels via qRT-PCR in the tardigradeHypsibius exemplaris

Abstract

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes.1 – 3 To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on transcriptional profiling4, 5 and RNA interference (RNAi)6 to define molecular targets. Such studies require efficient, accurate, and robust RNA extraction methods; however, obtaining high-quality quantitative levels of RNA from H. exemplaris has been challenging6, 7. Possessing a layer of firm chitinous cuticle, tardigrade tissues are difficult to disrupt by chemical or mechanical means8. Here we present an efficient single-tardigrade, single–tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but dramatically reduces the time required for extraction. We show that this RNA extraction method yields robust quantities of cDNA and can be used to amplify multiple transcripts by qRT–PCR. To validate the method, we use it to compare dynamic changes in expression of genes encoding two heat-shock-regulated proteins, Heat–Shock Protein 70 β2 (HSP70 β2) and Heat-Shock Protein 90 α ; (HSP90 α ;) by quantifying their expression levels in heat–exposed and cold–exposed individuals using qRT–PCR across long-term and short–term heat stressors. Our method effectively complements existing bulk RNA extraction methods7, permitting rapid examination of individual tardigrade transcriptional data and quantification of phenotypic variations in expression profiles amongst individuals.