We want to acknowledge the NIH Ruth Kirschstein Fellowship # 5F32AG081056-02 and the Errett Fisher Post-Doctoral Fellowship, which supported Dr. Molly J. Kirk, the Crowe Family Fellowship, which supported Chaoming Xu, and a University of California, Santa Barbara Academic Senate Grant, and NIH grants #R01GM143771 and #2R01HD081266, which supported these research efforts. The authors also acknowledge the use of the Biological Nanostructures Laboratory within the California NanoSystems Institute, supported by the University of California, Santa Barbara, and the University of California, Office of the President.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66935/66935fig01.jpg" /> 
Figure 1: Single-tube pipeline for RNA extraction from a single tardigrade. (A)&#160;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)&#160;Image of micropipette taper used for removal of water. Scale bar: 2 mm. (C)&#160;Bright field image of a tardigrade in a small volume of water (dotted line). Removal of most water to the extent shown is required for successful extraction and prevents dilution of lysis buffer. Scale bar: 50 &#956;m. (D)&#160;Image showing immersion of samples in liquid nitrogen using long forceps to rapidly freeze-thaw the samples safely. Some of the content was created in BioRender. Kirk, M. (2022) BioRender.com/d93s511 <a href="https://www.jove.com/files/ftp_upload/66935/66935fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: Figure 1A shows a schematic of the procedure. For detailed tardigrade and algal culturing procedures, refer to previously published reports45,46,47.
1. Sterilization of spring water
<ol>
	<li>Pour 2 L of spring water from a 5-gallon water jug (see Table of Materials for Specifics) into a 2 L autoclave-safe glass bottle.</li>
	<li>Place the cap on the autoclave-safe bottle and seal with a small amount of autoclave tape. Do not tighten the bottle; place the cap on top.</li>
	<li>Autoclave the spring water for 50 min on a wet cycle with no drying step.</li>
	<li>Allow the water to come to room temperature (RT), and seal the cap firmly before storing it at RT.</li>
</ol>
2. Glass micropipette pulling (with a pipette puller)
<ol>
	<li>Secure a glass micropipette (O.D.: 1 mm, I.D.: 0.58 mm, Length: 10 cm) on a micropipette puller. Avoid contact with the heating filament, as this will alter the pipette shape and damage the filament.
	<ol>
		<li>Determine the pulling of the pipette empirically for each filament and pipette puller. However, to serve as a starting point for optimization, use 78 &#176;C and a single pull step of pull weight of 182.2 g.</li>
	</ol>
	</li>
	<li>Allow the filament to heat and gravity to separate the glass micropipette into two glass micropipettes with sharp points (Figure 1B).</li>
	<li>Store these pulled glass micropipettes in a closed 100 mm Petri dish with wax or clay to hold them in place and prevent the sharp tips from breaking.</li>
</ol>
3. Glass micropipette pulling (without a pipette puller)
<ol>
	<li>Light a Bunsen burner or other controlled flame source on a low setting.</li>
	<li>Take a glass micropipette with one end in each hand.</li>
	<li>Hold the center of the glass micropipette over the flame until the glass begins to melt. Then, rapidly pull the two ends apart. This will create two very delicate sharp tips.</li>
	<li>Lightly break the tip with a pair of sterile fine forceps.</li>
	<li>Store these pulled glass micropipettes in a closed 100 mm Petri dish with wax or clay to hold them in place and prevent the sharp tips from breaking.</li>
</ol>
4. RNA extraction
<ol>
	<li>Obtain 0.5 L of liquid nitrogen in a cryo-safe container. 
	CAUTION: Liquid nitrogen is cryogenic and may cause burns if exposed to skin or eyes. When handling, use protective clothing, splash goggles, nitrile gloves, cryo-gloves, a lab coat, and closed-toed shoes. Ascertain that the container is liquid nitrogen safe before transporting the liquid. Using an ethanol-dry ice bath for this step may also be possible.</li>
	<li>Make cDNA synthesis master mix: a 10 &#181;L solution containing 1 &#181;L of random hexamer primer, 2 &#181;L of DNase, 4 &#181;L of 5x RT Buffer, 1 &#181;L Enzyme Mix, 1 &#181;L of H2O, and 1 &#181;L of 10 mM dNTPs. Store this solution on ice.</li>
	<li>Prepare tardigrade lysis buffer (5 mM Tris (pH = 8), 0.5% (v/v) Detergent 1, 0.5% (v/v) Detergent 2, 0.25 mM EDTA in sterile nuclease-free Water). 
	NOTE: This solution can be stored on the bench top for 6 months. However, maintain sterility and avoid potential RNAse-contaminating sources.</li>
	<li>Aliquot enough lysis buffer for extractions (2 &#956;L/ tardigrade).</li>
	<li>Add RNAse inhibitor to the tardigrade lysis buffer solution to a final concentration of 4 U/&#956;L.</li>
	<li>Vortex and spin down the solution at RT on a bench-top centrifuge at a speed of 2000 x g for 5 s before storing the solution on ice.</li>
	<li>Remove as many tardigrades as needed for the experiment from culture using a sterile filter-tipped P1000 pipette and place them in a sterile 35 mm Petri dish. 
	NOTE: Any number of tardigrades may be processed in this way. Usually, three tardigrades per condition are processed for extraction.</li>
	<li>Wash the tardigrades three times, using 1 mL of autoclaved sterile spring water and a sterile filter-tipped P1000 pipette. Slowly pipetting them up and down helps to remove algal contaminants.</li>
	<li>Using a dissecting microscope at 25x to 50x magnification, transfer a single tardigrade from this washed culture to a new sterile 35 mm Petri dish using a sterile filter-tipped P10 pipette.</li>
	<li>Use a sterile filter-tipped P200 pipette to wash the single tardigrade in 100 &#956;L of sterile nuclease-free water. 
	NOTE: This wash step is used to further remove contaminants, including ribonucleases.</li>
	<li>Transfer the washed tardigrade to the bottom of a clean, sterile PCR tube in 1-2 &#956;L of sterile nuclease-free water using a sterile filter-tipped P10 pipette, carefully ensuring the tardigrade does not stick to the side of the tip.</li>
	<li>Visualize the tardigrade under a dissecting microscope at 25x magnification.</li>
	<li>To facilitate water removal of water, break the tip of the pulled glass micropipette lightly outside of the tube. Ensure the bore is big enough to pull up the water but not the tardigrade.</li>
	<li>Using the capillary action of a pulled glass micropipette, remove water until the animal is surrounded by a small bubble of water approximately two tardigrade lengths in diameter.</li>
	<li>Monitor the water removal process via the dissecting scope to ensure the water level is appropriate and the tardigrade remains hydrated. 
	NOTE: Figure 1C offers an example of how much water to remove. This is a critical step. A small bubble of water will surround the tardigrade to prevent it from drying out, but as much excess water as possible should be removed to prevent dilution of the lysis buffer. For an example of the remaining water levels, please refer to Figure 1C.</li>
	<li>Immediately after removing the water, add 2 &#956;L of tardigrade lysis buffer to the bottom of the tube, briefly vortex, and centrifuge the tube at RT for 5 s at 2000 x g on a tabletop centrifuge.</li>
	<li>Immediately place the samples containing the tardigrades into a PCR tube rack and ensure that they are held tight by the rack.</li>
	<li>Grip the rack using a pair of long coarse forceps and gently dip the rack containing the samples into the liquid nitrogen until fully frozen (Figure 1D).</li>
	<li>Remove the rack from the liquid nitrogen and immediately place it on ice. Allow the sample to thaw (takes ~45 s to 1 min). Monitor the sample every 15 s by removing it from the ice and visibly inspecting it. Once the sample is visibly transparent, move on to the next step.</li>
	<li>Repeat steps 4.18-4.19 five more times. A total of six freeze-thaw cycles are required for maximal lysis and extraction (Figure 2A,B).</li>
	<li>Once the freeze-thaw is complete, place samples on ice and immediately progress to the next step. Do not freeze the samples at this point for storage, as this will diminish available RNA for cDNA preparation.</li>
</ol>
5. cDNA synthesis
<ol>
	<li>Add 2 &#956;L of cDNA synthesis master mix to the PCR tube containing tardigrade lysate. Briefly flick the tube and spin it at RT at 2000 x g for 5 s with a tabletop centrifuge before replacing the samples on ice.</li>
	<li>Place the samples in a thermocycler and incubate at 25 &#176;C for 10 min to anneal primers, at 55 &#176;C for 30 min to perform reverse transcription, and finally, heat-inactivate enzymes at 85 &#176;C for 5 min.</li>
	<li>After the incubation, immediately place the tube on ice and dilute the sample to a total volume of 25 &#956;L by adding 21 &#956;L of sterile nuclease-free water. For low-copy number transcripts, alter this dilution step as determined empirically.</li>
</ol>
6. qPCR
<ol>
	<li>Determine the annealing temperature of the primer set using total RNA prepared from larger amounts of tardigrades, for example, the bulk extraction method presented in Boothby, 201843.</li>
	<li>Run a PCR temperature gradient to determine the optimal annealing temperature before running qRT-PCR (for all PCR settings used in this protocol, refer to Table 1 and Table 2).</li>
	<li>Thaw one tube of indicator dye super mix on ice and isolate from light. Place a 96-well qPCR plate on ice and place 5 &#956;L of super mix, 2 &#956;L of water, 1 &#956;L of each primer (10 &#956;M), and 1 &#956;L of cDNA product in the number of desired wells.</li>
	<li>Seal the PCR plate with plate seal and run the qRT-PCR using an annealing temperature appropriate for the primer set (for all qRT-PCR settings used in this paper, refer to Table 3).</li>
</ol>
7. Quantification and results interpretation
<ol>
	<li>Compare the results quantitatively to one or more control housekeeping genes, whose expression is expected to be constant over the imposed conditions. For this study, the actin gene was used.</li>
	<li>Obtain and compare the Ct-values or cycle threshold for each well to the Ct values of the control housekeeping gene reactions. Calculate the fold change in gene expression using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/66935/66935eq01.jpg" style="margin: auto;" /> 
	<img alt="Equation 2" src="/files/ftp_upload/66935/66935eq02.jpg" style="margin: auto;" /> 
	<img alt="Equation 3" src="/files/ftp_upload/66935/66935eq03.jpg" style="margin: auto;" /> 
	NOTE: Fold gene expression is plotted for each transcript and tardigrade as a 2-(&#916;&#916;Ct)48.</li>
	<li>To obtain a rough estimate of the transcript number from the Ct-value, use the following equation: 
	<img alt="Equation 4" src="/files/ftp_upload/66935/66935eq04.jpg" style="margin: auto;" /> 
	Where N is the number of transcripts, and 2 is the assumed PCR efficiency or the fold increase in fluorescence per cycle of PCR48.</li>
</ol>

Optimized RNA Extraction for qRT-PCR in Individual Tardigrades

The authors declare no conflicts of interest to disclose.

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 &gt;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...

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&#160;that is lethal to humans2,3,4,5,6,7,8,9,10, nearly complete desiccation11,12,13,14,15, freezing in the absence of added cryoprotectants16,17,18, and, in their desiccated state, even the vacuum of space19,20. Owing to their unique capacity for survival in extreme environments, these animals have become foundational models for understanding extremotolerance in complex, multicellular organisms1,21,22,23.
Stable genetic manipulation of these remarkable animals, including transgenesis and germline gene modification, has remained elusive until recently24,25. As such, most experiments to reveal molecular mechanisms of extremotolerance are performed through transcriptional profiling via RNA sequencing. Many valuable and informative RNA sequencing data sets exist for tardigrades under various extreme conditions, ranging from radiation8,9,26,27,28, heat stress29, freezing stress12, and desiccation27,30,31,32,33. Some of these studies have utilized bulk RNA extraction and purification methods to illuminate our molecular understanding of extremotolerance. However, bulk extraction of RNA transcripts from many animals prevents analysis of variation in gene expression between individuals, thus missing the potential richness of more refined data sets. Importantly, these studies often analyze heterogeneous populations of animals that include both animals that survive environmental stressors and those that do not. As such, these studies are confounded by averaging expression data from multiple and potentially dramatically different response states. To address this issue, Arakawa et al., 201634 developed an elegant low-input RNA-seq pipeline that applies an RNA extraction kit followed by a linear PCR amplification step using single34,35,36 or&#160;multiple30,37,38animals as input. These studies have been foundational to our understanding of tardigrade extremotolerance22. Interestingly, this protocol has also been applied to qRT-PCR using seven animals as starting material24.
In most model organisms, having identified potential targets via RNA-seq, qRT-PCR is then performed to confirm transcriptional changes identified by RNA-seq and assess the expression time course of candidate genes in a high-resolution manner. To test the function of identified genes, such studies are often followed by RNAi-mediated knockdown of molecular targets39,40 and analysis of extremotolerant capacity12,41. The efficacy of each RNAi knockdown is typically confirmed by qRT-PCR by directly monitoring the decrease in transcript abundance. However, RNAi is a labor-intensive process in tardigrades as each dsRNA must be delivered via manual microinjection of individuals39,40. Owing to the low throughput nature of this strategy, a rapid, low-cost RNA extraction method adapted for qRT-PCR from single animals would be highly valuable for tardigrade research. Although previous methods have been developed to extract RNA from single tardigrades, these protocols have not combined their extraction with qRT-PCR, instead relying on optical density-based methods12,40,41. Motivated by these challenges, we sought to develop a protocol that reliably yields RNA in quantity and quality that can be used for qRT-PCR from single H. exemplaris.
Adapted from a single-animal RNA extraction protocol developed for Caenorhabditis elegans42, STST is optimized for H. exemplaris. The extraction method consists of six rapid freeze-thaw steps, physically disrupting the cuticle, allowing RNA extraction and subsequent cDNA synthesis. The STST method decreases extraction time by more than 24-fold compared to bulk RNA extraction methods, as described by Boothby, 201843, and by 30% compared to single tardigrade RNA extraction kits, as described by Arakawa et al., 201634. Further, the number of sample-experimenter interactions is decreased from 5 to only 1 compared to RNA extraction kit preparations, thus reducing the risk of contamination by exogenous ribonucleases. When querying for highly expressed genes, the STST method produces sufficient cDNA for 25 quantitative RT-PCR reactions per single tardigrade, requiring only 1 &#956;L of the total 25 &#956;L cDNA volume per reaction. However, template concentrations need to be empirically determined for lower abundance transcripts.
The efficacy of the STST method for analyzing dynamic changes in gene expression was evaluated by investigating the differential expression of the genes encoding heat-shock protein-90&#945; (HSP90&#945;) and heat-shock protein 70&#946;2 (HSP70&#946;2) in response to short-term heat-shock at 35&#176;C for 20 minutes. Both HSP70&#946;2 and HSP90&#945; in most eukaryotic organisms are rapidly upregulated following short-term heat-shock exposure (20 min)42. Analysis in H. exemplaris revealed that both the HSP70&#946;2 and&#160;HSP90&#945;-encoding RNAs extracted from single heat-treated tardigrades showed statistically significant increases in expression following short-term heat exposure. These findings demonstrate that the STST protocol can be used to analyze dynamic changes in gene expression in individual animals over time.
The STST extraction method should complement existing experimental methods such as RNA-seq by facilitating rapid and inexpensive RNA extraction and subsequent comparison of transcript levels by qRT-PCR. This method will also be valuable for assessing the efficiency and penetrance of RNAi in manually injected individuals more quantitatively than optical density alone. Finally, owing to their similar cuticular structures and physical characteristics, it is likely that this method will also be effective for analyzing gene expression in other tardigrade species44.

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

single-animal, single-tube rna extraction for comparison of relative transcript levels via qrt-pcr in the tardigrade hypsibius exemplaris

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 &#946;2 (HSP70 &#946;2) and Heat-Shock Protein 90&#945; (HSP90&#945;), 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.

Development and optimization of single-tardigrade RNA extraction 
Adapting the protocol from Ly et al., 201542 for RNA extraction in tardigrades, the STST system is optimized to maximize the quantity and quality of the preparation (Figure 1A). RT-PCR was performed for actin transcripts, quantifying transcript yield by amplifying a 527 bp region spanning exons 1 and 2 (sequences for these primers can be found in Table 1). The opti...

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

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).

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular ...

single-animal-single-tube-rna-extraction-for-comparison-relative

Research

JoVE Journal

Biology

308 Views.  University of California, Santa Barbara. 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). 

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

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Environment

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Genetics

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunology and Infection

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

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

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Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Expression Analysis of Mammalian Linker-histone Subtypes

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

In situ Hybridization for Sipunculus nudus Coelomic Fluid

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Laser Microdissection for Species-Agnostic Single-Tissue Applications