We thank Sharlene Murdoch and the Babraham Institute Facilities for their support. JLP was supported by the Wellcome Trust (093970/Z/10/Z) and OC is supported by ERC 638426 and BBSRC [BBS/E/B000C0426].

NOTE: Throughout this protocol Caenorhabditis elegans is referred to as &#8220;worm&#8221; or &#8220;worms&#8221;. A variety of C. elegans strains can be ordered through online databases or by directly contacting labs that use the model organism. Part I of this protocol (sections 1&#8722;3) describes cDNA preparation through the Worm-to-CT protocol. Part II of this protocol (sections 4&#8722;13) describes running high-throughput RT-qPCR using nanofluidics, adapted from a protocol developed by Fluidigm16. This protocol applies to the use of the two types of nanofluidic chips defined earlier, the single-array chip, which can monitor 96 targets into 96 samples (9,216 RT-qPCR reactions total), or the multi-array chip, which functions as subunits of 12 target x 12 samples. Every multi-array chip contains six independent arrays that can be run together or separately. For instance, using a whole multi-array chip can monitor 72 targets x 12 samples (or vice versa), or 36 targets x 24 samples (or vice versa). For further information regarding any of the materials used in this protocol, refer to the Table of Materials.
1. RT-qPCR primer validation
NOTE: Real-time primers were designed based on the recommended properties originally issued by MIQUE guidelines17. To make primers specific for processed RNA, products were designed such that the two primers bound to either side of at least one splice junction. Requirements for suitable primers included 20%&#8722;80% guanine and cytosine content, a melting temperature of 58&#8722;60 &#176;C, a difference in melting temperature between primer pairs of &#8804;0.5 &#176;C and a product length of 70&#8211;120 bp. The sequence of the primers generated can be found in Supplemental Table 1. Open source code for the scripts used to generate the primers can be found at https://github.com/s-andrews/wormrtpcr. Primer pairs for transcripts with splice sites were designed so that they lie in two exons flanking an intron but were not designed to be splice-variant specific, using NCBI Primer Blast software18. For this study, the primer sets were blasted against the C. elegans genome to test for any off-target complementarity.
<ol>
	<li>Retrieve primers from the database of RT-qPCR primers (Supplemental Table 1). Alternatively, design qPCR primer pairs using online tools such as NCBI Primer Blast18.</li>
	<li>Perform a qPCR standard curve to monitor specificity and PCR efficiency for each pair of primers using standard bulk qPCR techniques19 and following MIQUE guidelines17,20. 
	NOTE: Only primer pairs with R2 &#62; 0.98 and PCR efficiency between 85% and 115% should be used. The sequence, PCR efficiency, and R2 for the primers used in this study are detailed in Table 3.</li>
</ol>
2. Worm lysis through Worm-to-CT
<ol>
	<li>Pick the worms from their bacterial lawn onto a fresh, unseeded NGM plate and allow the worms to move around the plate for 5 min to remove most of the bacteria from the worm through its movement. 
	NOTE: The bacterial lawn and growing conditions will differ depending on the experimental design. The experiments presented here require 6 cm NGM plates seeded with OP50 Escherichia coli with worms of interest grown to stage L4.9 in a 20 &#176;C incubator.</li>
	<li>In an RNase-free hood, prepare a master mix consisting of 12.5 &#181;L of 2x RT buffer, 1.25 &#181;L of 20x RT enzyme buffer, and 0.25 &#181;L of nuclease-free water per sample. Add 14 &#181;L of master mix to 11 &#181;L of each sample from step 2.8.</li>
	<li>Place the lid of a PCR strip upside down on the platform of the dissecting scope and add 10 &#181;L of the lysis mix to domed PCR tube caps under a compound microscope. 
	NOTE: With only one or two samples, it is better to use a PCR strip containing at least four tubes, as this reduces the risk of the caps blasting open in the subsequent freeze-thaw steps. Alternatively, rubber bands can be used to hold the caps in place. In that case ensure caps are properly closed every time the tubes are transferred.</li>
	<li>Pick the worms from the plate into each slot of the lid containing the lysis mix by &#8220;scooping&#8221; them (i.e., catching the worms underneath with the pick) to avoid bacterial contamination. Close the tubes and spin them down for 5 s using a tabletop microcentrifuge (Table of Materials) before placing them in a Dewar flask filled with liquid nitrogen. 
	NOTE: Between 15 and 30 worms should be used for bulk experiments and 1 worm for single-worm measurements. 
	CAUTION: When handling liquid nitrogen wear Cryo-Gloves as well as protective eyewear and adhere to standard clothing regulations, because contact with the skin or eyes can cause serious frostbite injury.</li>
	<li>Freeze-thaw the PCR tubes 10x by transferring them between liquid nitrogen and a ~40 &#176;C water bath. Leave the tubes in the liquid nitrogen for a minimum of 5 s to ensure the samples are completely frozen. Leave the tubes in the water bath until the samples thaw. Do not leave in for a longer period, as this leads to RNA degradation. 
	NOTE: Tubes can be left in liquid nitrogen for an extended period of time, as samples are frozen to approximately -200 &#176;C, reducing RNA degradation. This should not, however, be a protocol stopping point, because liquid nitrogen evaporates rapidly.</li>
	<li>Mix the samples on a thermal mixer (Table of Materials) set at 4 &#176;C for 20&#8722;30 min rotating at ~1,800 rpm.</li>
	<li>While the samples are being mixed, thaw the stop solution on ice.</li>
	<li>Spin the samples down using a tabletop microcentrifuge (Table of Materials)&#160; and add 1 &#181;L of stop solution to each tube. 
	NOTE: The samples can be left at -80 &#176;C for up to 1 week before reverse transcribing the RNA (section 3).</li>
</ol>
3. Reverse transcription
NOTE: For reverse transcription of single worms, the results shown here were generated using the reagents provided with the nanofluidic chips (option 2 in Figure 1). The reagents highlighted in option 2 of Figure 1 were also used for reverse transcription of pooled samples. Either method works interchangeably for the different sample types.
<ol>
	<li>Reverse transcription of single worms
	<ol>
		<li>In an RNase-free hood, add 1.25 &#181;L of reverse transcription mix (Table of Materials) to a fresh PCR tube. 
		NOTE: A 96 well PCR plate and an automatic pipette can be used if there are many samples. The manufacturer&#39;s protocol states that 1 &#181;L can used per sample.</li>
		<li>Take 5 &#181;L of the lysis solution and stop solution mix from step 2.8 and add it to the fresh PCR tube containing the reverse transcription mix. 
		NOTE: The manufacturer&#39;s protocol states that 1 &#181;L of RNA (2.5 pg/&#956;L&#8211;250 ng/&#956;L) can be used per reaction. A negative RT control per plate can be added by replacing the reverse transcription mix with 5 &#181;L of lysed sample and 1.25 &#181;L of RNAse-free water.</li>
		<li>Run the samples using the following reverse transcription program on a thermocycler: 25 &#176;C for 5 min, 42 &#176;C for 30 min, 85 &#176;C for 5 min and 4 &#176;C for &#8734;. 
		NOTE: The cDNA produced can be stored at -20 &#176;C before proceeding to amplification and data collection using high-throughput qPCR.</li>
	</ol>
	</li>
	<li>Reverse transcription for bulk samples (15&#8722;30 worms)
	<ol>
		<li>In an RNase-free hood, prepare a master mix consisting of 12.5 &#181;L of 2x RT buffer, 1.25 &#181;L of 20x RT enzyme buffer, and 0.25 &#181;L of nuclease-free water per sample. Add 14 &#181;L of master mix to 11 &#181;L of lysis solution and stop solution mix from step 2.8. 
		NOTE: When dealing with a large number of samples this can be performed in 96 well plates.&#160;</li>
		<li>Run the samples through a thermocycler using the following reverse transcription program: 37 &#176;C for 60 min, 95 &#176;C for 5 min, and 4 &#176;C for &#8734;.</li>
		<li>Dilute the produced cDNA 1:4 in nuclease-free water. 
		NOTE: Generally, the products come to a final volume of 25 &#181;L, in which case 75 &#181;L should be added. However, due to condensation the final volume can vary. Therefore, adjust accordingly to make a 1:4 ratio in the final solution. This dilution step does not apply if performing qPCR on single worms. The cDNA produced can be stored at -20 &#176;C before proceeding to amplification and data collection using high-throughput qPCR.</li>
	</ol>
	</li>
</ol>
4. Preparing the multiplex primer mix
<ol>
	<li>Prepare a forward/reverse (F/R) primer stock for each pair of primers at 50 &#181;M final concentration. Mix the same volume of forward and reverse primers at 100 &#181;M each.</li>
	<li>Combine 1 &#181;L of 50 &#181;M F/R primer stock for each primer pair to be tested. Add the DNA suspension buffer up to a total volume of 100 &#181;L. 
	NOTE: The stock primer concentrations here differ from those described in the manufacturer&#8217;s protocol16&#160;but retain the same final concentration of 500 nM.</li>
</ol>
5. Target specific preamplification
<ol>
	<li>Prepare a master mix containing 1 &#181;L of preamplification mastermix (Table of Materials), 0.5 &#181;L of the pooled primer mix (step 4.2), and 2.25 &#181;L of nuclease-free water per reaction with a 10% overall surplus volume.</li>
	<li>In a 96 well plate, aliquot 3.75 &#181;L of the master mix into as many wells as required for the number of samples to be run.</li>
	<li>Add 1.25 &#181;L of the cDNA solutions of interest generated at step 3.1.3 or 3.2.3 to each well.</li>
	<li>Cover the plate with 96 well sealing tape, briefly vortex, and centrifuge with a tabletop plate spinner. Transfer to a thermocycler and run the following program: 95 &#176;C for 2 min, 15 cycles of denaturation at 95 &#176;C for 15 s, annealing/extension at 60 &#176;C for 4 min, and 4 &#176;C for &#8734;. 
	NOTE: The manufacturer recommends from 10&#8722;20 cycles for the preamplification reaction16. This protocol recommends 10 or 15 cycles depending on the expression levels of the target genes.</li>
</ol>
6. Exonuclease I treatment
NOTE: This is to remove unincorporated primers from preamplification.
<ol>
	<li>Prepare an exonuclease I mix containing 0.2 &#181;L of exonuclease I reaction buffer (Table of Materials), 0.4 &#181;L of exonuclease I at 20 U/&#181;L (Table of Materials), and 1.4 &#181;L of nuclease-free water per sample. Keep all reagents on ice, especially the exonuclease I.</li>
	<li>Remove the 96 well plate (step 5.4) from the thermocycler, centrifuge with the tabletop plate spinner, and carefully remove the seal. Add 2 &#181;L of the exonuclease I mix to each preamplification reaction. Reseal, centrifuge, and place the 96 well plate back into the thermocycler using the following program: 37 &#176;C for 30 min, 80 &#176;C for 15 min, and 4 &#176;C for &#8734;.</li>
	<li>Take the samples out of the thermocycler and dilute them 1:5 by adding 18 &#181;L of 1x Tris EDTA buffer (Table of Materials). 
	NOTE: It is possible to keep the cDNA samples at -20 &#176;C for later use. The manufacturer&#8217;s protocol suggests potential dilutions of 5x, 10x, or 20x at this stage16, depending on the expression level of the targets of interest.</li>
</ol>
7. Preparing the assay mixes
NOTE: Assay mixes can be prepared in 384 well plates, as the wells have the same spacing as the nanofluidic chips, making loading easier.
<ol>
	<li>Preparing assay mixes for a multi-array chip
	<ol>
		<li>Prepare a master mix consisting of 2 &#181;L of 2x assay loading reagent (Table of Materials) and 1.6 &#181;L of DNA suspension buffer (Table of Materials) for each well according to the prepared plan. Aliquot 3.6 &#181;L of this master mix per well into a 384 well plate.</li>
		<li>Add 0.4 &#181;L of the 50 &#181;M F/R primer stock prepared in step 4.1 to the appropriate wells according to the prepared plan. 
		NOTE: This provides a total of 4 &#181;L of assay mix per well, with a surplus of 1 &#181;L.</li>
	</ol>
	</li>
	<li>Preparing assay mixes for a single-array chip
	<ol>
		<li>Prepare a master mix consisting of 3 &#181;L of 2x assay loading reagent and 2.4 &#181;L of DNA suspension buffer for each well according to the prepared plan. Aliquot 5.4 &#181;L of this master mix per well into a marked 384 well plate.</li>
		<li>Add 0.6 &#181;L of the 50 &#181;M F/R primer stock to the appropriate wells according to the prepared plan. 
		NOTE: This provides a total of 6 &#181;L of assay mix per well, with a surplus of 1 &#181;L.</li>
	</ol>
	</li>
</ol>
8. Preparing the sample mixes
NOTE: Sample mixes can be prepared up to 1 day in advance and stored at 4 &#176;C.
<ol>
	<li>Preparing samples for a multi-array chip
	<ol>
		<li>Prepare a sample master mix consisting of 2 &#181;L of 2x fluorescent probe supermix with low ROX (Table of Materials) and 0.2 &#181;L of sample reagent (Table of Materials) per sample. Dispense 2.2 &#181;L of this mix into the marked 384 well plate. 
		NOTE: The manufacturer recommends not vortexing the sample reagent16.</li>
		<li>Pipette 1.8 &#181;L of each preamplified, exonuclease I treated sample from step 3.2.3 into the appropriate wells according to the prepared plan. 
		NOTE: This gives a total of 4 &#181;L, with a surplus of 1 &#181;L.</li>
	</ol>
	</li>
	<li>Preparing samples for a single-array chip
	<ol>
		<li>Prepare a sample master mix consisting of 3 &#181;L of 2x fluorescent probe supermix with low ROX (Table of Materials) and 0.3 &#181;L of 20x DNA-binding dye sample loading reagent (Table of Materials) per sample. Dispense 3.3 &#181;L of this mix into the marked 384 well plate.</li>
		<li>Pipette 2.7 &#181;L of each preamplified and exonuclease I treated sample from step 6.3 into the appropriate wells according to the prepared plan. 
		NOTE: This gives a total of 6 &#181;L, with a surplus of 1 &#181;L. If there are any wells to be run without a sample these must be loaded with sample master mix and 2.7 &#181;L of water instead of cDNA. This is recommended for both chip types. The machine needs low ROX in every inlet in order to detect the chip&#8217;s grid.</li>
	</ol>
	</li>
</ol>
9. Priming the nanofluidic chip
NOTE: A multi-array chip only needs to be primed on the first run. If there are subsequent runs of the same chip this stage can be skipped. These steps are the same for both chip types.
<ol>
	<li>Slowly and carefully, inject the full 150 &#181;L of the control line fluid from the syringes included into the accumulators of the chip. Ensure that no control liquid touches the chip by holding the chip at a 45&#176; angle and holding the tip of the syringe away to avoid spillage.</li>
	<li>Remove the blue protective film from the bottom of the chip.</li>
	<li>Place the chip into the nanofluidics PCR priming machine (Table of Materials), with the barcode facing outwards. Run the &#8216;Prime (153x)&#8217; script, which takes ~15&#8211;20 min. 
	NOTE: Turn on the nanofluidics thermocycler (Table of Materials) at this stage, because the camera takes about 10 min to cool down to below 0 &#176;C.</li>
</ol>
10. Loading the nanofluidic chip
<ol>
	<li>Remove the barrier plugs sequentially as loading takes place. This reduces the chance of misloading wells.</li>
	<li>Transfer either 3 &#181;L for a multi-array chip or 5 &#181;L for a single-array chip of each primer assay mixes and sample mixes to the corresponding inlets of the nanofluidic chip according to the prepared plan. Make sure not to introduce bubbles, which can cause the total volume transferred to be smaller than desired. 
	NOTE: If there are any wells that will be run without a primer it is important for these to be loaded with master mix, substituting the primer volume with water. This applies to both chip types. At this stage, it can be easier to place the chip on a dark surface, which will allow the wells to be seen more easily.</li>
</ol>
11. Running the nanofluidic chip
NOTE: The first time running a multi-array chip, set up the tracking file by selecting Tools | Flex Six Usage Tracking, click New, enter a file name, and select a location before clicking Done.
<ol>
	<li>Open the data collection software. Click Start New Run. Place the loaded chip into the nanofluidics thermocycler with the barcode facing outwards.</li>
	<li>Choose the project setting if applicable, then click Next | Load. If loading a multi-array chip, select the partitions (arrays) to be run.</li>
	<li>Select the application Reference Probes, then change the application type to Gene Expression, and change the passive reference to ROX. Select the Single Probe Assay, change the probe type to Eva Green, and click Next.</li>
	<li>Select the thermal cycling protocol GE FLEX six Fast PCR+Melt v1 to run a multi-array chip, or the protocol GE 96.96 Fast PCR+Melt v2 to run a single-array chip.</li>
	<li>Confirm that Auto Exposure is selected and click Start Run.</li>
</ol>
12. Post chip run
NOTE: This section is only necessary for multi-array chips when not using the entire chip.
<ol>
	<li>Take the chip out of the nanofluidics thermocycler and load into the nanofluidics PCR priming machine and run the Post Run (153x) script, which lasts 5 min.</li>
	<li>Label the plugs used for personal reference. 
	NOTE: The chip can now be stored at room temperature and the remaining arrays on the chip can be run within 2 months.</li>
</ol>
13. Data cleanup and analysis
<ol>
	<li>Open the data in the &#8216;Real-Time PCR Analysis&#8217; software (Table of Materials). Check the melting peak temperature for every primer pair tested. Eliminate results exhibiting more than one melting temperature peak, for a given primer pair. 
	NOTE: Multiple peaks only appear occasionally, presumably when primer pairs forms dimers, or from interactions of target primers with other primers in the pooled primer mix.</li>
	<li>Export the data as a &#8216;heatmap&#8217; spreadsheet file and eliminate failed samples or primers.</li>
	<li>Analyze data using the standard Delta-Ct method21. For statistical evaluation perform one-way ANOVA on relative expression levels.</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotypic+Signals+from+Neural+HSF-1+Separate+Thermotolerance+from+Longevity.">Heterotypic Signals from Neural HSF-1 Separate Thermotolerance from Longevity.</a> Cell Reports. 12 (7), 1196-1204 (2015).</li><li>Zhang, X., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dbl-1+a+TGF-beta+is+essential+for+C.+elegans+aversive+olfactory+learning.">Dbl-1 a TGF-beta is essential for C. elegans aversive olfactory learning.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (42), 17081-17086 (2012).</li><li>S&#248;rensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+and+Misuse+of+the+Coefficient+of+Variation+in+Organizational+Demography+Research.">The Use and Misuse of the Coefficient of Variation in Organizational Demography Research.</a> Sociological Methods &amp; Research. 30 (4), 475-491 (2002).</li><li>Pujol, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+innate+immune+responses+to+infection+and+wounding+in+the+C.+elegans+epidermis.">Distinct innate immune responses to infection and wounding in the C. elegans epidermis.</a> Current Biology. 18 (7), 481-489 (2008).</li><li>Brennecke, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accounting+for+technical+noise+in+single-cell+RNA-seq+experiments.">Accounting for technical noise in single-cell RNA-seq experiments.</a> Nature Methods. 10 (11), 1093-1095 (2013).</li><li>Ly, K., Reid, S. J., Snell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+RNA+analysis+of+individual+Caenorhabditis+elegans.">Rapid RNA analysis of individual Caenorhabditis elegans.</a> MethodsX. 7 (2), 59-63 (2015).</li></ol>

The authors have nothing to disclose.

In this paper, the Worm-to-CT protocol is shown to be a rapid and efficient method to extract RNA from single worms or a small pool of worms. The high-throughput offered by the nanofluidic system makes it ideal for quantification of inter-individual variability measurements. Furthermore, the high sensitivity of this method allows the detection of genes expressed at low levels that fall below detection when using single-worm RNA-seq technologies9.
When considering the choice of method to prepare cDNA from single worms. Ly et al.29 optimized a protocol that relies on proteinase K for cuticle digestion. The cuticle is a major hurdle for the isolation of molecules from worms and proteinase K provides an effective method to break it. However, proteinase K has to be heat-inactivated to be able to use enzymes for reverse transcription. While Ly et al. used a 10 min exposure to 96 &#176;C, this step was avoided in this protocol because RNA is easily degradable. Instead of using proteinase K, repeated freeze-thaw cycles were used to break the cuticle. The freeze-thaw is an effective method to break the cuticle because more RNA can be isolated per worm. Ly et al. report that the total RNA extracted per worm is 35 ng using proteinase K, whereas this protocol obtains 51.75 ng &#177; 6.74 SEM of total RNA per worm. Avoidance of heat exposure coupled with preamplification steps apparently widens Worm-to-CT&#8217;s dynamic range of detection compared to standard protocols. Ly et al. report absolute Ct values of 21.1 &#177; 0.15 for hsp-16.2 and 22.8 &#177; 0.17 for hsp-70 after heat shock. Using the same heat shock conditions (1 h at 30 &#176;C), this protocol obtains absolute Ct values of 17.93 &#177; 0.57 for hsp-16.2 and 21.13 &#177;0.33 for hsp-70. This indicates that the freeze-thaw lysis method provides higher yields of RNA and is more appropriate for lowly expressed transcripts.
Nanofluidic systems are ideal when investigating a given set of target transcripts and the use of either smaller (multi-array chip) or larger (single-array chip) number of samples allowing adaptation to the scale of the experiment. To obtain an unbiased picture of all transcripts expressed in a single worm, the obvious choice is to use RNA sequencing. If, however, the focus of the experiment is a smaller but still relatively large set of target genes, it is more cost-effective to utilize this protocol, provided the researcher has access to a nanofluidics PCR machine. The cost of the nanofluidic system reagents and a single-array chip is estimated as approximately &#163;13 per worm, whereas the costs of the reagents for single-worm sequencing would be approximately &#163;60 per worm, not including the sequencing costs.
When considering what PCR platform to use, the Worm-to-CT method coupled to nanofluidic qPCR offers advantages with regards to time and throughput. It is possible to obtain 9,216 RT-qPCR results in approximately 2 days of work, whereas amplification of the same number of targets using a standard qPCR platform would take approximately 5 working weeks using 96 well plate assays, running four plates a day. However, if the number of targets to be tested is smaller, then it is more cost-effective to use Worm-to-CT coupled with a standard qPCR machine. The single-array chips cannot be rerun, so running empty wells decreases cost-efficiency.
One limitation of the method is the potential formation of primer-primer dimers during the multiplexing step, but this occurs in less than 1% of the cases. Although the Worm-to-CT protocol is efficient and provides reliable results when applied to single worms, there is a failure rate of about 5%, which likely corresponds to cases where the worm remains trapped in the cap or the top of the tube during the harvesting step.
Together, this versatile and reliable method offers increased throughput and sensitivity compared to more standard techniques. This method can be very useful for validation of high-throughput screens and is an excellent choice to either monitor or validate single-worm gene expression levels. This method can be applied to other challenging techniques, such as the quantitation of gene expression from isolated tissues. For example, isolation of full tissues, such as the intestine, gonads, or cells isolated by FACs, provides enough material to perform RNA sequencing experiments. However, limited amounts of material often lead to duplicated reads, which precludes quantitation of rare transcripts. In this scenario, using nanofluidics-based technology should provide added sensitivity to the experiments and increase cost-efficiency if the researchers need to monitor only a subset of all transcripts in those tissues or cells.

The optimization of single-cell RNA sequencing and qPCR revealed that transcriptional pulses or bursts can lead to massive variation in the number of RNA molecules per cell1. Further, these technologies uncovered substantial cellular heterogeneity previously missed by standard bulk transcriptomic measurements. Depending on the context, some single-cell transcriptional variability is caused by mixed cellular composition of tissues. However, even in isogenic cell populations grown under the same environment there is widespread transcriptional heterogeneity2,3. This &#8216;biological variability&#8217; is increasingly identified as a ubiquitous property of cellular networks, from bacteria to man. In some cases, it can have phenotypic consequences in development, cancer progression, HIV latency, and response to chemotherapy4,5.
The nematode Caenorhabditis elegans is a unique model organism with ideal characteristics for studying the causes and consequences of biological variability between individuals. These nematodes are a simple model organism composed of 959 cells, and their transparent cuticle makes them amenable for in vivo imaging studies6. C. elegans is a hermaphroditic species that predominantly reproduces through self-fertilization; this resulted in isogenic laboratory strains. Despite isogenicity and controlled culture conditions, many phenotypes and transcripts are variable across individuals, suggesting that stochastic or microenvironmental differences contribute to heterogeneity across individuals7,8. Such variability in gene expression has multiple fitness consequences, including variability in the penetrance of mutations, survival, developmental timing, and fecundity7,8,9. Due to these features, single-worm studies provide the unprecedented opportunity to study biological variability in a whole organism.
There is a fundamental need in the field to develop and optimize technologies for accurate detection of transcripts at a single-worm level. New technologies, such as single-worm RNA sequencing10, RNA sequencing from isolated tissues11, and single-cell sequencing12 are now available for C. elegans. However, a main challenge remains: when monitoring interindividuality, weakly expressed genes often fall below detectable levels13. This is particularly relevant for rare transcripts isolated from small amounts of starting material, as there is a well-established, inverse relationship between mean expression and technical variance, often causing rare transcripts to fall below statistical cutoffs13. The optimization of high-throughput multiplexed qPCR technologies has proven useful for mammalian single-cell studies, in particular when studying the expression of rare transcripts14,15. This technology can also be used for benchmarking and validation purposes of other single-worm technologies.
Worm-to-CT is a fast, robust method adapted from a kit used in cell biology studies, for single-worm cDNA preparation. cDNA obtained by this method coupled with multiplexed nanofluidic qPCR technology was chosen because it provides higher experimental throughput, a broader dynamic range of detection and has been validated for single-cell purposes14,15. The cDNA preparation described is also applicable for use with standard PCR technologies. The throughput is increased in two ways: First, cDNA preparation is faster and more reliable than traditional guanidium thiocyanate-phenol-chloroform extraction, because worms are directly added to the lysis buffer, skipping direct isolation of easily degradable RNA. Second, utilizing nanofluidic technologies significantly increases the number of samples and targets that can be run simultaneously. In this paper, two chips are compared: a single-array chip and a multi-array chip. A single-array chip can run 96 single worms and 96 primer sets, resulting in 9,216 reactions per experiment. To accomplish a similar throughput using standard qPCR technologies would require 96 separate qPCR experiments, using 96 well plates. The smaller and more flexible multi-array chip consists of six 12 x 12 arrays resulting in 864 reactions. The method&#8217;s superior reliability and sensitivity are boosted by nanofluidic technology and by the introduction of a preamplification step. The method presented in this paper is meant to be used together with a state-of-the art statistical algorithm to extract biological variance. This article presents the protocol for rapid cDNA preparation and high-throughput qPCR for both single-worm and batch worm samples; the algorithm will be published elsewhere. For this protocol, the organization of each chip should be prepared prior to the experiment. Table 1 and Table 2 show examples of these plans for a multi-array and single-array chip, respectively. There are also overviews of the Worm-to-CT protocol detailed in Figure 1 and running the multi-array and single-array chips in Figure 2.

<table><tbody><tr><td>100&amp;micro;M stock primers for genes of interest.</td><td></td><td></td><td></td></tr><tr><td>20X DNA Binding Dye</td><td>Fluidigm</td><td>100-7609</td><td>for 96.96 chip (Single-Array Chip)</td></tr><tr><td>2X Assay Loading Reagent</td><td>Fluidigm</td><td>100-7611</td><td></td></tr><tr><td>384 well plates</td><td></td><td></td><td></td></tr><tr><td>8-strip PCR tubes</td><td></td><td></td><td></td></tr><tr><td>96 well ice block</td><td></td><td></td><td></td></tr><tr><td>96 well plates</td><td></td><td></td><td></td></tr><tr><td>96.96 Dynamic Array IFC</td><td>Fluidigm</td><td>BMK-M-96.96</td><td>Referenced throughout the manuscript as &quot;Single-Array Chip&quot;</td></tr><tr><td>96-well sealing tape</td><td></td><td></td><td></td></tr><tr><td>BioMark &amp;amp; EP1 Software</td><td>Fluidigm</td><td>101-6793</td><td>Contains: Biomark HD Data Collection software, Real-Time PCR Analysis software, SNP Genotyping Analysis software, Digital PCR Analysis software, Melt Curve Analysis software</td></tr><tr><td>BioMark or BioMark HD system</td><td>Fluidigm</td><td>100-2451 K1</td><td>Referenced throughout the manuscript as &quot;nanofluidics thermocycler&quot;</td></tr><tr><td>Control Line Fluid Kit for 96.96 and FlexSix IFCs</td><td>Fluidigm</td><td>89000021</td><td>Referenced throughout manuscript as &quot;control line fluid&quot;</td></tr><tr><td>DNA Suspension buffer</td><td>TEKnova</td><td>T0021</td><td></td></tr><tr><td>domed PCR caps</td><td></td><td></td><td></td></tr><tr><td>Exonuclease I</td><td>New England BioLabs</td><td>M0293L</td><td></td></tr><tr><td>Exonuclease I reaction buffer</td><td>New England BioLabs</td><td>B0293S</td><td></td></tr><tr><td>FLEXsix DELTAgene Sample Reagent</td><td>Fluidigm</td><td>100-7673</td><td>referenced throughout manuscript as &quot;sample reagent&quot;</td></tr><tr><td>FLEXsix Gene Expression IFC</td><td>Fluidigm</td><td>100-6308</td><td>referenced throughout manuscript as &quot;Multi-Array Chip&quot;</td></tr><tr><td>Fluidigm Real-Time PCR Analysis User Guide</td><td>Fluidigm</td><td>68000088</td><td>This is the protocol used as a basis for section 2 of the protocol, referenced within the manuscript.</td></tr><tr><td>IFC Controller MX</td><td>Fluidigm</td><td>68000112 I1</td><td>Referenced throughout the manuscript as &quot;nanofluidics PCR priming machine&quot;</td></tr><tr><td>IFC Controllers SOFTWEAR</td><td>Fluidigm</td><td>100-2297</td><td>Contains all softwear and scripts required for running the IFC controller MX</td></tr><tr><td>liquid nitrogen in dewar</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>StarLab</td><td>C1301B-230V</td><td>Used to spin down PCR tube strips in the protocol.</td></tr><tr><td>Nuclease Free Water</td><td></td><td></td><td></td></tr><tr><td>plate spinner</td><td>Labnet</td><td>K4050725</td><td>mini plate spinner, mps 1000. referenced through the protocol as &quot;tabletop plate spinner&quot;</td></tr><tr><td>Power SYBR Green Cells-to-Ct kit</td><td>invitrogen</td><td>4402955</td><td>This kit is that which is adapted for use in nematodes in the Worm-to-CT protocol. Contense: Store Stop Solution, DNase I and 20X RT Enzyme Mix at -20&amp;deg;C. Store Lysis Solution, 2X SYBR RT Buffer (referenced throughout the manuscript as RT buffer), and Power SYBR&lt;sup&gt;&amp;reg;&lt;/sup&gt;Green PCR Master Mix (refferenced througout the manuscript as PCR Master Mix). This Kit is for 400 reactions, kits also available for 40 and 100 reactions.</td></tr><tr><td>PreAmp Master Mix</td><td>Fluidigm</td><td>100-5580, 100-5581</td><td></td></tr><tr><td>Reverse Transcription Master Mix&amp;mdash;480 reactions</td><td>Fluidigm</td><td>100-6299</td><td>One tube also available for 96 reactions (PN100-6298)</td></tr><tr><td>Rnase Free water</td><td></td><td></td><td></td></tr><tr><td>SsoFast EvaGreen Supermix with Low ROX</td><td>Bio-Rad Laboratories</td><td>172-5211</td><td>referenced throughout the manuscript as &quot;fluorescent probe supermix&quot;</td></tr><tr><td>Standard 96 and 384-well Thermal Cycler</td><td></td><td></td><td></td></tr><tr><td>TE Buffer (10mM Tris, pH8.0, 1.0mM EDTA</td><td>TEKnova</td><td>T0224</td><td>Referenced throughout the manuscript as Tris-EDTA buffer</td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000031</td><td>Referenced throughout the text as &quot;thermal mixer&quot;</td></tr><tr><td>Trizol</td><td>Thermo-Fisher</td><td>15596026</td><td>Referenced through the protocol as &quot;guanidium thiocyanate-phenol-chloroform&quot;</td></tr><tr><td>Warm water bath</td><td></td><td></td><td></td></tr></tbody></table>

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.

Validation of Worm-to-CT as a cDNA preparation method
To test if the Worm-to-CT protocol is a valid cDNA extraction method, it was compared to standard guanidium thiocyanate-phenol-chloroform extraction methods. The results are shown in Figure 3, where cDNA was prepared from an average of ~1,000 worms using standard guanidium thiocyanate-phenol-chloroform extraction techniques22 and from 30 worms using the Worm-to-CT method. The samples were heat shocked simultaneously (30 min at 34 &#176;C). Globally, hsp-70 mRNA expression levels per 100 ng of total RNA were comparable using both methods. However, in the case of highest hsp-70 expression (i.e., in N2 following heat shock) expression levels were higher with the Worm-to-CT method, indicating improved sensitivity.
To determine if an expected decrease in hsp expression in hsf-1(sy441)23, a mutation in the main transcriptional regulator of molecular chaperones23,24, could be reproduced, transcriptional chaperone induction following a brief heat shock was compared. With both methods a decrease in hsp-70 induction was detected in hsf-1(sy441) animals. This was expected, because mutant hsf-1(sy441) animals exhibit a decreased ability to induce chaperones due to a truncation in the transactivation domain of HSF-1. For guanidium thiocyanate-phenol-chloroform extraction hsp70 decreased by 82.7% compared to controls and 92.3% for Worm-to-CT compared to wild type animals (Figure 3). The results were comparable between both methods and comparable to previous reports23. These results indicate that the Worm-to-CT method is a valid alternative to standard cDNA synthesis techniques.
Validation of the nanofluidics PCR platform used to amplify mRNA targets
To test the consistency of the results using nanofluidic qPCR for transcript amplification, the PCR results obtained from the Worm-to-CT bulk method were compared on both a standard qPCR system (Table of Materials) and a nanofluidic qPCR system using a multi-array chip. The fold change in the expression of three different genes, sma-3 (Figure 4A), sma-10 (Figure 4B), and dnj-26, was monitored (Figure 4C) in animals carrying a null allele in dbl-1 (dbl-1(nk3))25 compared to wild type counterparts. Dbl-1 encodes the sole ligand of the Bone Morphogenetic Protein (BMP) signaling pathway. sma-3 and sma-10 are genes encoding SMAD orthologues, key components of the BMP signaling cascade. Dnj-26 encodes a molecular chaperone, a target of BMP signaling. These results show little to no difference in the fold change comparing the results of the two methods, resulting in not significant P-values at 0.3113, 0.2635, and 0.3481 for sma-3, sma-10, and dnj-26, respectively. Altogether, these results show that the Worm-to-CT method applied to bulk samples is an efficient and rapid way to extract RNA from few worms and provides reliable data when coupled with either standard PCR systems or high-throughput nanofluidics-based qPCR platforms.
Comparison between the expression levels obtained by bulk samples with averages obtained from single worms
The relative expression levels were calculated using either cDNA obtained from bulk samples (25 worms) or from an average of 36 single worm samples (Figure 5). Both cDNAs were obtained using the Worm-to-CT method and amplified using nanofluidics PCR technology. As observed in Figure 5A&#8211;C, for all chaperones tested (i.e., hsp16.1, F44E5.4, hsp-70), the methods detected comparable expression levels. These results indicate that parameters obtained from single worms are reliable.
Application of Worm-to-CT coupled to nanofluidics technology to estimate single-worm gene expression parameters
Because the single-array chip allows monitoring of up to 96 target transcripts on 96 individual samples, it is therefore well-suited to monitor individual variability in transcript expression between single worms. Figure 6A presents a representative result showing the mean expression of multiple hsp transcripts from single worms following a short heat shock. As observed in the figure, the variability in the expression of transcripts differed dramatically across different genes (Figure 6A). To gain further insight, the coefficient of variation (CV) was calculated by dividing the standard deviation by the mean of the expression levels26 (Figure 6B). Three genes whose CV values have been previously estimated by alternative methods were monitored (unpublished data). Two stable transcripts (ife-1 and Y45F10D.4) and one variable (nlp-2927) showed their expected variability. The graph also clearly depicts the well-known inverse relationship between variability values and expression levels26 (Figure 6B).
Technical replicates are of paramount importance to ensure reproducibility when using bulk samples. However, this is not necessarily the case for single-cell experiments14,15,28. To determine if the use of technical replicates is necessary for parameter estimation when using single-worm samples, 28&#160;individual worms were harvested,&#160;following a short heat shock, and processed using technical triplicates. The CV values calculated from single-worm data obtained in triplicate (blue dots in Figure 7, technical CV) versus those for every transcript obtained from individual worms (red dots in Figure 7, biological variability) were compared. For every transcript tested, the technical CVs were lower than the biological CVs, indicating that technical triplicates were not required for parameter estimation. The fact that technical replicates are not required increases the throughput of the experiment without compromising quality.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61132/61132fig01.jpg" /> 
Figure 1: Overview of the Worm-to-CT Protocol. 
This figure shows a brief overview of the different steps required to run worms through the Worm-to-CT protocol. Two optional methods are shown for the reverse transcription step; these are interchangeable methods for either type of chip. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61132/61132fig02.jpg" /> 
Figure 2: Overview of the preparation and running of nanofluidic qPCR. 
This figure depicts preparations for running the nanofluidic qPCR system using a multi-array chip and a single-array chip. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61132/61132fig03.jpg" /> 
Figure 3: Worm-to-CT protocol on bulk samples provided reliable results. 
Comparison of Worm-to-CT protocol versus regular guanidium thiocyanate-phenol-chloroform extraction22 on bulk samples. Consistent with previous findings, in hsf-1(sy441) mutants23, the levels of hsp transcripts in response to heat shock decreased. The above histograms depict the induction of hsp-70 in the absence of (-), or following (+) a short heat shock of 30 min at 34 &#176;C. The cDNA was obtained using guanidium thiocyanate-phenol-chloroform extraction applied to 1,000 worms (left) or using the Worm-to-CT method applied to 30 pooled worms (right). The expression levels of hsp-70 per 100 ng of total RNA obtained by each method were compared. As expected, in hsf-1(sy441) the transcriptional induction of hsp-70 in response to heat shock significantly decreased by 82.7% using guanidium thiocyanate-phenol-chloroform and by 92.3% using the Worm-to-CT method. The mRNA levels from target genes were normalized against the average of the three housekeeping genes cdc-42, pmp-3, and ire-1. Each dot represents a biological replicate. Data were log transformed for statistical analysis, as they did not meet the conventions required for parametric analysis. Statistical analysis was done using a RM-One-way ANOVA using Sidak&#8217;s multiple comparisons test. Wild type = N2, hsf-1 = hsf-1(sy441). Bars denote the standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61132/61132fig04.jpg" /> 
Figure 4: Expression patterns were consistent between standard qPCR and nanofluidic qPCR systems. 
(A) The expression level of sma-3&#160;(A), sma-10 (B) or dnj-26 (C) mRNA was determined through regular qPCR and nanofluidic qPCR (multi-array chip) from three biological replicates of cDNA generated through Worm-to CT from the wild type strain (N2) and the dbl-1(nk3) knockout strain25. Relative mRNA expression levels were determined for each strain using the Delta-Ct method21. Fold change was then determined by dividing the expression levels obtained in dbl-1(nk3) worms by the corresponding mRNA levels in the N2 strain. As shown in panel A, the patterns were consistent for both methods in each individual biological replicate. (B) and (C) are the same as (A) for sma-10 and dnj-26 mRNA levels, respectively. Target mRNA levels were normalized against the housekeeping genes cdc-42 and pmp-3. Statistical analysis was calculated for each gene using a paired t-test comparing the results of the three biological replicates produced through standard qPCR and those generated through nanofluidic qPCR. The P-values of these comparisons were 0.3113, 0.2635, and 0.3481 for sma-3, sma-10, and dnj-26, respectively. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61132/61132fig5v2.jpg" /> 
Figure 5: Using Worm-to-CT method on bulk samples or on single worms provided similar levels of expression when normalized per worm. 
The expression levels of (A) hsp-16.1/11, (B) F44E5.4, and (C) hsp-70 (C12C8.1) were analyzed in young adult animals in the absence of heat shock either by performing Worm-to-CT on a bulk of 25 animals, or on 36 single individuals. When the data were normalized per worm, there was no significant difference between levels obtained per worm for each transcript using both methods. The mRNA levels from target genes were normalized against the average of the three housekeeping genes cdc-42, pmp-3, and ire-1. Bars represent the standard error of the mean. Statistics = paired t-test. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61132/61132fig06.jpg" /> 
Figure 6: High-throughput RT-qPCR on single worms using the Worm-to-CT method could monitor inter-individual variability in gene expression. 
(A) The mean expression levels for 53 transcripts obtained upon exposure to a short heat shock (30 min at 34 &#176;C). Boxplots represent the distribution of mean mRNA expression from individual worms (an average of three technical replicates were used per individual worm). The dots represent expression levels in 28 individual worms. The mRNA levels from target genes were normalized against the average of the three housekeeping genes cdc-42, pmp-3, and ire-1. (B) The coefficient of variation26 (CV) as a function of mean mRNA expression for 53 transcripts following exposure to a short heat shock was calculated from 28 individual animals (raw data shown in panel B). The set of transcripts includes the variable nlp-29 transcript27 and two stable transcripts (ife-1 and Y45F10D.4; unpublished data). The CV is the ratio of the standard deviation to the mean. This CV was utilized to estimate inter-individual variability in transcript expression between individual worms. As expected, inter-individual variability scaled with decreased mean expression levels. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61132/61132fig07.jpg" /> 
Figure 7: Technical replicates were not necessary when analyzing inter-individual variability in gene expression using a nanofluidic chip. 
The data presented in this graph were obtained in 28 individual worms following a short heat shock (30 min at 34 &#176;C). Each red dot represents the coefficient of variation (CV) of mean transcript expression levels for one transcript assayed between 28 individual worms (bio CV). Each blue dot represents the CV of expression levels between three technical replicates obtained from a single worm, per transcript assayed (technical CV). This graph shows that technical variability (between technical replicates) was much lower than biological variability (between individual worms), suggesting that it is unnecessary to perform technical replicates on a nanofluidic gene expression array when assaying gene expression in single worms, similarly to single-cell studies14,15,28. <a href="https://www.jove.com/files/ftp_upload/61132/61132fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Plan layout for a multi-array chip. The table above shows a simple layout that can be utilized when planning a multi-array chip run. On the left are the spaces that should be filled with the primer targets of interest and on the right are spaces that should be filled with the samples of interest. Each assay and sample array is paired number-wise through the chip. <a href="https://cloudflare.jove.com/files/ftp_upload/61132/Table_1.xlsx" target="_blank">Please click here to download this table.</a>
Table 2: Plan layout for a single-array chip. The table above shows a simple layout that can be utilized when planning a single-array chip run. On the left are spaces that should be filled with primer targets of interest and on the right are spaces that should be filled with the samples of interest. <a href="https://cloudflare.jove.com/files/ftp_upload/61132/Table_2.xlsx" target="_blank">Please click here to download this table.</a>
Table 3: List of RT-qPCR primers used in this study. <a href="https://cloudflare.jove.com/files/ftp_upload/61132/Table_3.xlsx" target="_blank">Please click here to download this table.</a>
Supplemental Table 1: Primers from the database of RT-qPCR primers. <a href="https://cloudflare.jove.com/files/ftp_upload/61132/Table_S1.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology at JoVE.com

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.

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 ...

high-throughput-quantitative-rt-pcr-single-bulk-c-elegans-samples

Research

JoVE Journal

Genetics

7.8K Views.  Babraham Institute. In dit artikel wordt een protocol met hoge doorvoer beschreven voor een snelle en betrouwbare bepaling van de genexpressieniveaus in enkele of bulk C. elegansmonsters. Dit protocol vereist geen RNA-isolatie en produceert cDNA rechtstreeks uit monsters. Het kan worden gebruikt samen met high-throughput multiplexed nanofluidic real-time qPCR platforms.

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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biology

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Immunology and Infection

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Source: Prior, H., et al. <a href="https://www.jove.com/video/57518/a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c">A Rapid and Facile Pipeline for Generating Genomic Point Mutants in&#160;C. elegans&#160;Using CRISPR/Cas9 Ribonucleoproteins.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.

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

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