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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;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&#160; Tips</td>
		</tr>
		<tr>
			<td>1000 &#181;L&#160; 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&#160; Tips</td>
		</tr>
		<tr>
			<td>200 &#181;L&#160; 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&#160; Tips</td>
		</tr>
		<tr>
			<td>4 Star Straight Strong Medium Point Tweezer&#160;</td>
			<td>Excelta&#160;</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&#160;3M LEAD FREE AUTOCLAVE TAPE 1&#34;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>NC0802040</td>
			<td>Refered to as Autoclave Tape&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>BioRadHard-Shell 96-Well PCR Plate</td>
			<td>BioRad&#160;</td>
			<td>HSS9641</td>
			<td>Refered to as PCR Plate&#160;</td>
		</tr>
		<tr>
			<td>BULWARK FR Lab Coat:&#160;</td>
			<td>Grainger&#160;</td>
			<td>26CF64</td>
			<td>Refered to as Lab Coat</td>
		</tr>
		<tr>
			<td>C1000 Touch Bio-rad Thermocycler&#160;</td>
			<td>BioRad</td>
			<td>1851148</td>
			<td>Refered to as Thermocycler&#160;</td>
		</tr>
		<tr>
			<td>C1000 Touch Bio-rad Thermocycler with CFX Optics Module&#160;</td>
			<td>BioRad</td>
			<td>1845097</td>
			<td>Refered to as qPCR thermocycler&#160;</td>
		</tr>
		<tr>
			<td>Chloroccoccum hypnosporum.&#160;&#160;</td>
			<td>Carolina</td>
			<td>152091</td>
			<td>Refered to as Algae</td>
		</tr>
		<tr>
			<td>Corning&#160;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 &#38; Company Vortex-Genie 2 Laboratory Mixer&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>3030A</td>
			<td>Refered to as Vortexer&#160;</td>
		</tr>
		<tr>
			<td>Direct-zol Micro Prep&#160;</td>
			<td>Zymo Research&#160;</td>
			<td>R2060</td>
			<td>Refered to as RNA extraction kit&#160;</td>
		</tr>
		<tr>
			<td>Dumont 5 Biology Tweezers</td>
			<td>Fine Science Tools&#160;</td>
			<td>11254-20</td>
			<td>Refered to as Fine Forceps</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Fisher Scientific</td>
			<td>S311-500&#160;</td>
			<td>Refered to as EDTA&#160;</td>
		</tr>
		<tr>
			<td>FIJI v 2.14.0/1.54f&#160;</td>
			<td>ImageJ,</td>
			<td>N/A</td>
			<td>Refered to as FIJI/ImageJ</td>
		</tr>
		<tr>
			<td>Filament for pippette Puller&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Glass Micropipette O.D. 1mm ID 0.58, Length 10 cm</td>
			<td>TriTech Research</td>
			<td>GD-1&#160;</td>
			<td>Reffered to as glass micropipette</td>
		</tr>
		<tr>
			<td>Hypsibius exemplaris Z151 Strain&#160;</td>
			<td>Carolina</td>
			<td>133960</td>
			<td>Refered to as Tardigrades&#160; or H. exemplaris&#160;</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen Dewar 1 L</td>
			<td>Agar Scientific&#160;</td>
			<td>AGB7475</td>
			<td>Refered to as Cryo-safe container&#160;</td>
		</tr>
		<tr>
			<td>Maxima H Minus First Strand cDNA Synthesis Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1651</td>
			<td>Refered to as cDNA Synthesis Master Mix&#160;</td>
		</tr>
		<tr>
			<td>Narishige Dual-Stage Glass Micropipette Puller&#160;</td>
			<td>Tritech Research</td>
			<td>&#160;PC-10</td>
			<td>Refered to as micropipette puller&#160;</td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>Fisher Scientific</td>
			<td>17-000-314</td>
			<td>Refered to as Nitrile Gloves&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>PIPETMAN P10, 1&#8211;10 &#181;L, Metal Ejector</td>
			<td>Gilson</td>
			<td>F144055M</td>
			<td>Refered to as P 10 Pipette</td>
		</tr>
		<tr>
			<td>PIPETMAN P1000, 100&#8211;1000 &#181;L, Metal Ejector</td>
			<td>Gilson</td>
			<td>F144059M</td>
			<td>Refered to as P 1000 Pipette</td>
		</tr>
		<tr>
			<td>PIPETMAN P200, 20&#8211;200 &#181;L, Metal Ejector</td>
			<td>Gilson&#160;</td>
			<td>F144058M</td>
			<td>Refered to as P 200 Pipette</td>
		</tr>
		<tr>
			<td>Pound This 4-Color Modeling Clay&#160;</td>
			<td>American Science Surplus</td>
			<td>&#160;96517P001</td>
			<td>Refered to as Clay&#160;</td>
		</tr>
		<tr>
			<td>Prism v10.0&#160;</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td>Refered to a Prism&#160;</td>
		</tr>
		<tr>
			<td>RNAse-Free, 8 Strip 0.2 mL PCR Tubes with caps&#160;</td>
			<td>Invitrogen</td>
			<td>AM12230</td>
			<td>Refered to as Sterile PCR Tube</td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitor</td>
			<td>Promega&#160;</td>
			<td>N2111</td>
			<td>Refered to as RNAse inhibitor&#160;</td>
		</tr>
		<tr>
			<td>Spring water&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Stereo-Microscope System w/optics and illumination&#160;</td>
			<td>TriTech Research</td>
			<td>SMT1</td>
			<td>Refered to as Dissecting Microscope</td>
		</tr>
		<tr>
			<td>Supertek Scientific&#160;Tirrill Burners</td>
			<td>Thermo Fisher Scientific</td>
			<td>S09572B</td>
			<td>Refered to as Bunsen Burner&#160;</td>
		</tr>
		<tr>
			<td>Table Top Centrifuge</td>
			<td>Qualitron</td>
			<td>&#160;DW-41-115-NEW</td>
			<td>Refered to as Table Top Centrifuge&#160;</td>
		</tr>
		<tr>
			<td>Tempshield&#160;Cryo-Gloves</td>
			<td>Fisher Scientific</td>
			<td>11-394-305</td>
			<td>Refered to as Cryo Gloves&#160;</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nunc Petri Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-757-099</td>
			<td>Refered to as 100 mm Petri dish&#160;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>T395-500&#160;</td>
			<td>Refered to as Tris or Tris Base&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka&#160;</td>
			<td>93443</td>
			<td>Refered to as Detergent 1&#160;</td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma aldrich</td>
			<td>P1379-500&#160;</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

269 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

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...