Multi-Species Tardigrade RNA Isolation Protocol

Summary

This protocol details RNA isolation procedures from various tardigrade species using a column-based RNA isolation kit designed to isolate RNA from small amounts of tissue and yield sufficient amounts of high-quality RNA for downstream applications such as RNA sequencing and complementary DNA synthesis.

Abstract

Tardigrades are microscopic animals that are known for their exceptional tolerance to environmental stress. These animals can survive desiccation, extreme pressure, low temperatures, and high levels of ionizing radiation. However, the molecular mechanisms underlying these extremotolerant capabilities remain incompletely understood. The molecular approaches used to investigate these mechanisms, which include RNA sequencing, RNA interference (RNAi), and heterologous expression of tardigrade genes in systems such as bacteria, require the isolation of high-quality messenger RNA (mRNA) from limited biological material. In this study, we describe a reproducible protocol for isolating total RNA from tardigrades that is (1) effective across multiple species, including a heterotardigrade species, (2) requires only 25–200 animals, depending on body size, and (3) yields RNA of sufficient quantity and quality for downstream applications. The present protocol successfully enables efficient RNA recovery from reasonable numbers of tardigrades and supports molecular investigations of stress tolerance across various tardigrade species.

Introduction

Tardigrades are well known for their ability to survive extreme stress that other animals would not, including desiccation, extreme pressures, extreme temperatures, and DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation^{1,2,3,4,5,6,7,8}. In recent years, scientists have investigated the mechanisms that underlie the extreme resilience of tardigrades using molecular approaches such as RNA sequencing, RNA interference (RNAi), and heterologous expression of tardigrade genes in other systems, including human cells and bacteria^{8,9,10,11,12,13,14,15,16,17,18,19,20}. When applied, these approaches have been a powerful tool to give us insight into how exactly tardigrades are capable of such extreme stress resistance^{8,9,10,11,12,13,14,15,16,17,18,19,20,21,22}. However, all of these molecular approaches to understanding tardigrade stress tolerance rely on the ability to isolate RNA from these tiny animals in ample quantity and with adequate quality for use in downstream processes.

A previous method for RNA isolation from tardigrades proved successful for researchers working with Hypsibius exemplaris²³. However, upon attempting to replicate this protocol in H. exemplaris, we found it to be unreliable in the resulting RNA concentration and quality (unpublished data). An alternative phenol-based RNA isolation method has been shown to work for H. exemplaris, Ramazzottius varieornatus, and a heterotardigrade species^{13,21,22,24,25,26}. This protocol has been shown to provide RNA of ample quality and quantity for downstream processes from both single animals and pools of animals (~50). However, this protocol requires the user to handle phenol-containing reagents. Phenol is a hazardous chemical that requires careful handling, and improper execution of this protocol can result in phenol carryover affecting RNA purity and downstream applications. Another previously published protocol for RNA isolation from tardigrades focuses on the ability to isolate RNA from single animals²⁷. Although this is a valuable protocol for applications such as quantitative PCR (qPCR)²⁷, it is less appropriate for downstream applications like RNA sequencing, where the goal is to attain a more holistic view of how animals respond to a certain stressor or condition.

These limitations in available RNA isolation protocols led to the investigation of alternative methods for isolating RNA from tardigrades. The column-based RNA isolation kit used in this protocol is marketed to isolate RNA from small amounts of tissue. This kit has also been used previously to isolate RNA from insect tissues for downstream RNA sequencing analysis, suggesting that it might be appropriate for isolations from other small, cuticle-bearing animals like tardigrades²⁸. This study outlines the step-by-step protocol for using this column-based RNA isolation kit with tardigrades, including explanations of specific cautions to be taken when performing this protocol with tardigrades. This article reports the success of this protocol in six tardigrade species (H. exemplaris, Milnesium sp., Paramacrobiotus experimentalis, Paramacrobiotus fairbanksi, Paramacrobiotus gadabouti, and the heterotardigrade Viridiscus celatus). The study has validated that RNA of substantial quality and quantity can be obtained from as few as 25 animals. Examples of resulting RNA concentration and quality data are also provided and are appropriate for downstream applications such as RNA sequencing (with minimal bias, exponential library amplification) and complementary DNA (cDNA) synthesis. This RNA isolation protocol has already proved useful to uncover mechanisms of radiation tolerance in H. exemplaris⁸. The success of this protocol across various tardigrade species suggests that it would be applicable more broadly to investigating stress-tolerance mechanisms, developmental mechanisms, and evolutionary processes.

Protocol

The following protocol does not require institutional animal ethics approval, as invertebrate animals are exempt from this requirement. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of equipment and area for RNA isolation

1. Turn on a heat block or water bath to 42 °C in preparation for RNA extraction.
2. Obtain a microfuge tube rack, pipettes (2 µL, 20 µL, 200 µL, and 1000 µL), and appropriate-sized filtered pipette tips.
3. Clean the benchtop, tube rack, pipettes, and tip boxes with RNase Away, dilute bleach solution (10%), or 70% ethanol to denature any residual RNase.
   NOTE: Excessive RNase on the equipment or in the area that you are isolating RNA may result in insufficient RNA quality after isolation due to degradation. Reduce this risk by allowing ample time for the RNase-denaturing chemicals to work, and by allowing the area and equipment to dry completely before proceeding.
4. Wear gloves for all steps of this protocol and frequently treat gloves with an RNase-denaturing solution throughout the protocol to prevent RNase degradation of RNA.

2. Collecting animals for RNA isolation

1. Using a pipette bulb attached to an aspirator tube assembly chamber fitted with a glass capillary (Figure 1A), an Irwin loop, or micropipette, move 25–200 animals (depending on species size) from their culture dishes to a dish with clean spring water to decrease the amount of non-tardigrade material (algae or other food sources like nematodes or rotifers, depending on species).
2. Obtain a 1.5 mL RNase-free tube and pestle.
3. Using a pipette bulb attached to an aspirator tube assembly chamber fitted with a glass capillary or an Irwin loop, move the animals into the bottom of the 1.5 mL RNase-free tube, taking special care to transfer as little water as possible with the animals.
4. Centrifuge at 4,500 g for 3 min to pellet the tardigrades into the bottom of the microfuge tube.
5. While observing the tube with pelleted tardigrades under a dissection microscope, use a 20 µL pipette or a pipette bulb attached to an aspirator tube assembly chamber fitted with a glass capillary with an opening smaller than the tardigrades you are isolating RNA from, to carefully remove as much water as possible without disturbing the pellet of tardigrades. See Figure 1B for a representation of an acceptable amount of remaining water.
   NOTE: Be cautious to leave a skim of water here so that the animals do not desiccate in the brief time between removal of water and addition of the Extraction Buffer (XB). If animals desiccate, this may result in poor RNA quality after isolation.
6. Add 100 µL of Extraction Buffer (XB) to the tube containing the tardigrades.

3. Mechanical disruption of tardigrades and RNA extraction

1. Using the RNase-free pestle, disrupt the tardigrades by gently but firmly grinding the pestle into the bottom of the tube for ~30 s. A total of 20–30 grinding actions should be sufficient to disrupt the tardigrades.
2. Remove the pestle from the tube, using caution to ensure that as much liquid as possible remains in the bottom of the tube.
3. Check the contents of the microfuge tube under the dissection microscope to ensure that there are no visible intact animals remaining.
4. Incubate the mixture at 42 °C for 30 min on a shaking heat block with gentle shaking (300 rpm).
   NOTE: The Extraction Buffer (XB) stabilizes the RNA to prevent degradation at this temperature.

4. Conditioning the RNA isolation column

1. In the last 10 min of the extraction incubation, assemble the column for RNA collection and purification. Place a purification column in a collection tube and label the column appropriately.
2. Add 250 µL Conditioning Buffer (CB) directly to the column.
3. Incubate at room temperature for 5 min.
4. Centrifuge at 16,000 × g for 1 min and discard flowthrough. The column is now ready to receive the RNA supernatant from the next step.
5. Remove the 10x DNase I Reaction Buffer and Nuclease-Free Water from the freezer to thaw completely in time for step 6 below.

5. Binding RNA to the column

1. After the extraction incubation, centrifuge the tube containing mechanically disrupted tardigrades and Extraction Buffer (XB) at 3,000 × g for 2 min.
2. Obtain a new RNase-free tube and label it accordingly.
3. Using a 200 µL pipette with a filter tip, carefully remove the supernatant from the pelleted tardigrade cuticles. Be careful not to disturb the cuticle pellet and to only remove supernatant.
   NOTE: Transferring cuticle into subsequent steps will result in a lower efficiency of column RNA yield due to physical clogging of the column. To avoid this potential error, you can observe the pipette tip with supernatant under the microscope to ensure the absence of cuticle. If any cuticle is present, put the supernatant back in the tube and repeat the centrifugation step.
4. Transfer the supernatant to your newly-labeled, RNase-free tube.
5. Pipette 100 µL 70% ethanol into the tube containing your RNA and mix thoroughly by pipetting up and down multiple times. At this step, one should see clear viscous filaments upon adding the ethanol. This is a good sign that there is RNA present in your supernatant.
6. Pipette the entire mixture (200 µL) onto your prepared column from step 4.
   NOTE: When pipetting any solutions onto the column for this and subsequent steps, take care to dispense the liquid into the middle of the column and avoid physically touching the column with the pipette tip. Mechanical puncture of the column will result in downstream issues with RNA quality and quantity.
7. To bind RNA to the column, centrifuge for 2 min at 100 × g, followed by centrifugation at 16,000 × g for 30 s to remove flowthrough.

6. Making DNase I solution

1. Allow the 10x DNase I Reaction Buffer and Nuclease-Free Water to thaw completely before making the solution.
2. For each RNA isolation, prepare a 40 µL solution in a separate 1.5 mL microfuge tube consisting of 32 µL Nuclease-Free Water, 4 µL 10x DNase I Reaction Buffer, and 4 µL DNase I Enzyme. Keep the solution on ice until ready to apply to the column in Step 7.3.

7. Washing the RNA and DNase I treatment

1. Pipette 100 µL Wash Buffer 1 (W1) directly onto the purification column containing the bound RNA (from step 5.7).
2. Centrifuge for 1 min at 8,000 × g and remove flowthrough.
3. Pipette 40 µL DNase I solution from Step 6 directly onto the purification column and incubate at room temperature for 15 min to remove DNA contamination.
4. Pipette 40 µL Wash Buffer 1 (W1) onto the purification column and centrifuge at 8,000 × g for 30 s.
5. Pipette 100 µL Wash Buffer 2 (W2) onto the purification column and centrifuge at 8,000 × g for 1 min.
6. Pipette another 100 µL Wash Buffer 2 (W2) onto the purification column and centrifuge at 16,000 × g for 2 min. Discard the flowthrough.
7. Centrifuge column at 16,000 × g for 1 min to dry the column.

8. Eluting RNA

1. Obtain a 0.5 mL tube (included in the kit) for each RNA isolation and label appropriately. This will be the tube that contains the RNA after isolation.
2. Transfer the column with bound and washed RNA to the labeled 0.5 mL tube. The column should click firmly into this tube.
3. While looking at the tube from the side, position the pipette tip directly above the column membrane, expel 11 µL of Elution Buffer (EB) onto the column, and incubate for 1 min at room temperature.
   NOTE: Failure to disperse the Elution Buffer (EB) directly onto the column will result in reduced RNA yields. If droplets of liquid are observed around the column perimeter, this suggests inappropriate dispersion of the Elution Buffer (EB) onto the column. If seen, it is recommended to repeat this step with the flowthrough from Step 8.5, ensuring that the Elution Buffer (EB) is expelled directly into the center of the column.
4. Place the 0.5 mL tube containing the column into the collection tube supplied with the kit for proper fit during centrifugation (Figure 2).
5. Centrifuge the column at 1,000 × g for 1 min to distribute the Elution Buffer (EB) and then spin at 16,000 × g for 1 min to elute the RNA.
6. Place the 0.5 mL tube containing the RNA on ice immediately.

9. Validation of RNA quantity and quality with a microvolume spectrophotometer

NOTE: Other methods can be used to assess RNA quantity and quality. Here, a method with a microvolume spectrophotometer is used. A fluorometer or microfluidics gel electrophoresis tool can also be used²⁹.

1. Wipe the pedestal of the microvolume spectrophotometer with a lint-free wipe to ensure it is clean of other liquids.
2. Select the RNA setting on the spectrophotometer for analysis.
3. Blank the spectrophotometer with 1 µL of the Elution Buffer (EB) used in Step 8.3.
4. After blanking, wipe the Elution Buffer (EB) off the pedestal with a lint-free wipe.
5. Keeping the 0.5 mL tube containing RNA on ice, pipette 1 µL of RNA onto the pedestal of the spectrophotometer. Lower the arm of the spectrophotometer to measure the absorbance of the sample to quantify and assess the quality of the isolated RNA.
   NOTE: Please see the “Results” section below for details about acceptable quality and quantity for downstream applications.

Results

A critical step in this protocol, as mentioned above, is removing an appropriate amount of water from the collected tardigrades prior to adding the Extraction Buffer (XB) for RNA extraction. Leaving too much water will result in dilution of the Extraction Buffer (XB) and in subpar (i.e., RIN < 7, 260/280 ratio <1.8, 260/230 ratio <1.8, and/or concentration <5 ng/µL) RNA extraction from the animals, resulting in reduced RNA concentration and quality after elution. Removing too much water will rapidly desiccate...

Discussion

The goal of this protocol is to facilitate the isolation of RNA from tardigrades that is of high enough quantity and quality to be applicable for downstream applications that facilitate investigations of tardigrade stress tolerance. The protocol presented here has outlined areas where users may encounter issues, including removing enough water prior to adding Extraction Buffer (XB) (Step 2.5), disrupting tardigrade bodies (Step 3.1–3.3), transferring the supernatant to the column for purification (Step 5.3), and el...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Microfuge Tubes	USA Scientific	1615-5500	Seal-Rite 1.5 mL graduated microcentrifuge tube, natural, polypropylene, 500/bag
70% Ethanol	VWR	71001-654	For cleaning benches/equipment
Arcturis PicoPure RNA Isolation Kit	Fisher	KIT0204	Kit containing components for the isolation protocol described in this manuscript
Centrifuge	Eppendorf	5405000441	Centrifuge 5425, rotary knobs, non-refrigerated, with Rotor FA-24x2, 120 V/50-60 Hz (US)
Deer Park Spring Water	Any Grocery Store	N/A	This is the media for tardigrade cultures and used to rinse tardigrades prior to RNA isolation
Dissection Microscope	Zeiss	N/A	Zeiss Stemi 305, zoom ratio 5:1 and total magnification 8x-40x, with transillumination Unit M LED
DNase I	Fisher	FEREN0521	Thermo Scientific DNase I, RNase-free (1 U/uL)
Filtered Pipette Tips 10 uL	VWR	76322-528	Any filtered pipette tip is fine
Filtered Pipette Tips 1000 uL	VWR	76322-154	Any filtered pipette tip is fine
Filtered Pipette Tips 20 uL	VWR	76322-134	Any filtered pipette tip is fine
Filtered Pipette Tips 200 uL	VWR	76322-150	Any filtered pipette tip is fine
Glass Capillaries	Fisher	50-821-811	World Precision Instrument Standard Glass Capillaries - 1B120-6 (6 inch, OD 1.2 mm, no filament). These get pulled into two glass capillaries with narrowed endings over a flame and then broken to the appropriate diameter to mouth pipette the tardigrade species of choice.
Heat Block	Eppendorf	5382000023	Eppendorf ThermoMixer C, basic device without thermoblock, 100-130 V/50-60 Hz (US/JP/South America/TW)
High Sensitivity RNA Screen Tape Ladder	Agilent	5067-5581	Ladder for high sensitivity analysis of total RNA on TapeStation Systems
High Sensitivity RNA ScreenTape	Agilent	5067-5579	For high sensitivity analysis of total RNA on TapeStation Systems
High Sensitivity RNA ScreenTape Sample Buffer	Agilent	5067-5580	Buffer for high sensitivity analysis of total RNA on TapeStation Systems
Microfluidics Gel Electrophoresis	Agilent	G2991BA	Agilent 4200 TapeStation System
Microvolume Spectrophotometer	Fisher	13400607	Thermo Scientific NanoDrop UltraC FL, Detector Type: 2048-element, CMOS Linear Image Sensor, Display: 10 in. High Definition Color Display, Languages: Chinese, English, French, Japanese, German, Light Source: Xenon Flash, Spectral Bandwidth: less than or equal to 1.8 nm (FWHM at Hg 254 nm), Voltage: 12 VDC
Mouth Pipette	Millipore Sigma	A5177	Aspirator tube assemblies for calibrated microcapillary pipettes
Nuclease Free Water	Fisher	FERR0581	Thermo Scientific Water, nuclease-free
Pipettes (2.5 uL, 20 uL, 200 uL, and 1000 uL)	Eppendorf	2231300004	Eppendorf Research plus, 4-pack
RNase Away	Fisher	21-402-178	Thermo Scientific RNase AWAY Surface Decontaminant
RNase-Free Tube and Pestle	Fisher	12-141-368	RNase-free tubes and pestles for RNA isolation