Single-Animal, Single-Tube RNA Extraction for Comparison of Relative Transcript Levels via qRT-PCR in the Tardigrade Hypsibius exemplaris

Summary

This work presents a rapid RNA extraction and transcript level comparison method for analyzing gene expression in the tardigrade Hypsibius exemplaris. Using physical lysis, this high-throughput method requires a single tardigrade as the starting material and results in robust production of cDNA for quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Abstract

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse transcription polymerase chain reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 β2 (HSP70 β2) and Heat-Shock Protein 90α (HSP90α), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR.

Introduction

Tardigrades are small multicellular animals renowned for their ability to survive extreme conditions that are lethal to most other forms of life¹. For example, these animals can survive nearly 1000 times the dose of ionizing radiation that is lethal to humans^{2,3,4,5,6,7,8,9,10}, nearly complete desiccation^11,

Protocol

Figure 1: Single-tube pipeline for RNA extraction from a single tardigrade. (A) Scheme showing the protocol for RNA extraction from a single tardigrade, including six freeze-thaw cycles and subsequent cDNA synthesis. Samples may subsequently be used for RT-PCR and qRT-PCR. (B) Image of micropipette taper used for removal of water. Scale bar: 2 mm. (C) Bright field image of a tardigrade in a....

Discussion

This study presents an efficient method for the extraction of RNA for single-tardigrade qRT-PCR. Directly comparing the STST methodology to an existing single tardigrade RNA extraction kit revealed that STST RNA extraction yields >200-fold higher amounts of actin RNA transcripts reduces the cost to less than one dollar per sample, and reduces the time required for extraction by 30%. To apply STST to a relevant biological question, we assessed the short-term heat-shock response expression profile. We found that transcr.......

Materials

Name	Company	Catalog Number	Comments
10 µL Premium Barrier Tips Low Binding, Racked, Sterile	Genesee Scientific	23-401	Refered to as Sterile Filter-Tipped P 10 Pipette  Tips
1000 µL  Premium Pipet Tips, Low Binding, Racked, Sterile	Genesee Scientific	23-165RS	Refered to as Sterile Filter-Tipped P 1000 Pipette  Tips
200 µL  Premium Barrier Tips Low Binding, Racked, Sterile	Genesee Scientific	23-412	Refered to as Sterile Filter-Tipped P 200 Pipette  Tips
4 Star Straight Strong Medium Point Tweezer	Excelta	00-SA-DC	Refered to as Long forceps
96-Well PCR Rack with Lid Assorted, 5 Racks/Unit	Genesee Scientific	27-202A	Refered to as PCR Rack
Andwin Scientific 3M LEAD FREE AUTOCLAVE TAPE 1"	Thermo Fisher Scientific	NC0802040	Refered to as Autoclave Tape
Autoclave Tape	Thermo Fisher Scientific	AB1170	Refered to as PCR Plate Seals
Benchling v8	Benchling	N/A	Refered to as Benchling
BioRadHard-Shell 96-Well PCR Plate	BioRad	HSS9641	Refered to as PCR Plate
BULWARK FR Lab Coat:	Grainger	26CF64	Refered to as Lab Coat
C1000 Touch Bio-rad Thermocycler	BioRad	1851148	Refered to as Thermocycler
C1000 Touch Bio-rad Thermocycler with CFX Optics Module	BioRad	1845097	Refered to as qPCR thermocycler
Chloroccoccum hypnosporum.	Carolina	152091	Refered to as Algae
Corning PYREX Reusable Media Storage Bottles	Thermo Fisher Scientific	06-414-1E	Refered to as 2 L Autoclave-safe Glass Bottle
Daigger & Company Vortex-Genie 2 Laboratory Mixer	Thermo Fisher Scientific	3030A	Refered to as Vortexer
Direct-zol Micro Prep	Zymo Research	R2060	Refered to as RNA extraction kit
Dumont 5 Biology Tweezers	Fine Science Tools	11254-20	Refered to as Fine Forceps
EDTA	Fisher Scientific	S311-500	Refered to as EDTA
FIJI v 2.14.0/1.54f	ImageJ,	N/A	Refered to as FIJI/ImageJ
Filament for pippette Puller	Tritech Research	PC-10H	Refered to as Filament
Fisherbrand Economy Impact Goggles	Fisher Scientific	19-181-501	Refered to as Splash Goggles
Glass Micropipette O.D. 1mm ID 0.58, Length 10 cm	TriTech Research	GD-1	Reffered to as glass micropipette
Hypsibius exemplaris Z151 Strain	Carolina	133960	Refered to as Tardigrades  or H. exemplaris
Liquid Nitrogen Dewar 1 L	Agar Scientific	AGB7475	Refered to as Cryo-safe container
Maxima H Minus First Strand cDNA Synthesis Kit	Thermo Fisher Scientific	K1651	Refered to as cDNA Synthesis Master Mix
Narishige Dual-Stage Glass Micropipette Puller	Tritech Research	PC-10	Refered to as micropipette puller
Nitrile Gloves	Fisher Scientific	17-000-314	Refered to as Nitrile Gloves
PETRI DISH, PS, 35/10 mm, WITH VENTS	Grenier	627102	Refered to as 35 mm Petri dish
PIPETMAN P10, 1–10 µL, Metal Ejector	Gilson	F144055M	Refered to as P 10 Pipette
PIPETMAN P1000, 100–1000 µL, Metal Ejector	Gilson	F144059M	Refered to as P 1000 Pipette
PIPETMAN P200, 20–200 µL, Metal Ejector	Gilson	F144058M	Refered to as P 200 Pipette
Pound This 4-Color Modeling Clay	American Science Surplus	96517P001	Refered to as Clay
Prism v10.0	GraphPad	N/A	Refered to a Prism
RNAse-Free, 8 Strip 0.2 mL PCR Tubes with caps	Invitrogen	AM12230	Refered to as Sterile PCR Tube
RNasin Ribonuclease Inhibitor	Promega	N2111	Refered to as RNAse inhibitor
Spring water	Nestle Pure Life	44221229	Refered to as Spring Water
SsoAdvanced Universal SYBR Green Supermix	BIO RAD	1725271	Refered to as Indicator Dye Super mix
Stereo-Microscope System w/optics and illumination	TriTech Research	SMT1	Refered to as Dissecting Microscope
Supertek Scientific Tirrill Burners	Thermo Fisher Scientific	S09572B	Refered to as Bunsen Burner
Table Top Centrifuge	Qualitron	DW-41-115-NEW	Refered to as Table Top Centrifuge
Tempshield Cryo-Gloves	Fisher Scientific	11-394-305	Refered to as Cryo Gloves
Thermo Scientific Nunc Petri Dishes	Thermo Fisher Scientific	08-757-099	Refered to as 100 mm Petri dish
Tris base	Fisher Scientific	T395-500	Refered to as Tris or Tris Base
Triton X-100	Fluka	93443	Refered to as Detergent 1
TWEEN 20	Sigma aldrich	P1379-500	Refered to as Detergent 2
Water - PCR/RT-PCR certified, nuclease-free	Growcells	PCPW-0500	Refered to as Sterile Nuclease Free Water