Single-animal, single-tube RNA extraction for quantitative analysis of transcripts in the tardigrade Hypsibius exemplaris .
| Autor: | Kirk MJ; Department of Molecular Cellular and Developmental Biology University of California, Santa Barbara, Santa Barbara, CA., Xu C; Department of Molecular Cellular and Developmental Biology University of California, Santa Barbara, Santa Barbara, CA., Rothman JH; Department of Molecular Cellular and Developmental Biology University of California, Santa Barbara, Santa Barbara, CA.; Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA. |
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| Jazyk: | angličtina |
| Zdroj: | BioRxiv : the preprint server for biology [bioRxiv] 2024 Mar 16. Date of Electronic Publication: 2024 Mar 16. |
| DOI: | 10.1101/2024.03.15.585302 |
| Abstrakt: | 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 profiling 4, 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 challenging 6, 7 . Possessing a layer of firm chitinous cuticle, tardigrade tissues are difficult to disrupt by chemical or mechanical means 8 . 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 β 2 (HSP70 β 2) 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 methods 7 , permitting rapid examination of individual tardigrade transcriptional data and quantification of phenotypic variations in expression profiles amongst individuals. Competing Interests: DISCLOSURES: Authors declare no conflicts of interest to disclose. |
| Databáze: | MEDLINE |
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