L.R.L. was funded by grants from the NIH/NIA (R00 AG042494 and R01 AG051810), a Glenn Foundation for Medical Research Award for Research in Biological Mechanisms of Aging and a Junior Faculty Grant from the American Federation for Aging Research. The authors would like to thank Anita Kumar and Shi Quan Wong for their useful feedback in the writing of this manuscript.

NOTE: Each macromolecule precipitation step is performed sequentially, followed by washes done concurrently; however, it is recommended to complete the RNA isolation first as it is intrinsically unstable.
1. Sample Collection
<ol>
	<li>Seed 1,000 worm eggs per 10 cm plate with appropriate growth conditions19. Incubate at 20 &#176;C for 72 h. 
	NOTE: Bleach egg-bearing adults in order to collect eggs as previously described19.</li>
	<li>Wash the plate with around 5 mL of M9 buffer and collect 1,000 adult worms into a tube. 
	NOTE: M9 buffer is composed of 35 mM sodium phosphate dibasic, 102 mM sodium chloride, 22 mM potassium phosphate monobasic, and 1 mM magnesium sulfate in sterile water19.</li>
	<li>Centrifuge the worms at 1,000 x g for 1 min, discard the supernatant, and transfer the pelleted worms with 1 mL of M9 buffer to a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge again at 845 x g for 1 min and discard most of the supernatant. Store the pelleted worms at -80 &#176;C for a minimum of 4 h. 
	NOTE: Using a frozen pellet produces a higher yield than a fresh pellet. A series of freeze/thaw cycles using liquid nitrogen or 95% ethanol on dry ice to crack the worm cuticle is recommended if using a fresh pellet. Leaving a small amount of M9 on the pellet will help break the cuticle when freezing.</li>
</ol>
2. Nucleotide and Protein Isolation
<ol>
	<li>Remove any supernatant from the thawed pellet and add 1 mL of cold GTCp reagent. Mix well by pipetting up and down. Place the sample on ice for 10 min and mix it occasionally by turning it upside down. 
	CAUTION: Phenol is corrosive, neurotoxic, and highly toxic and may cause chemical burns and blindness. 
	NOTE: We used up to 5,000 adult worms as the starting material with the reported volumes; each volume is based on the use of 1 mL of GTCp reagent. When using a larger sample, it may be necessary to scale up.</li>
	<li>To the solution of worms and GTCp, add 200 &#181;L of cold chloroform. Hold the tube between fingers and shake vigorously for 15 s. Leave it at room temperature (RT) for 3 min. 
	CAUTION: Chloroform is a toxic irritant, may cause skin sores and other organ-targeted harm, and is a possible carcinogen.</li>
	<li>Centrifuge the tube at 13,500 x g for 15 min at 4 &#176;C. 
	NOTE: Three layers are formed after this spin: the top, clear aqueous phase, the bottom, pink organic phase, and the small, cloudy interphase that contains lipids and DNA.</li>
	<li>Using a micropipette, move the clear top layer (aqueous phase) to a new RNase-free 1.5 mL microcentrifuge tube (tube A) to isolate RNA via alcohol precipitation (as detailed in step 2.5) and move the pink layer (organic phase) from the remaining pellet to a new tube (tube B) and place it on ice. 
	NOTE: The pink organic phase can be frozen at -80 &#176;C until the isolation of DNA and protein from this sample. Larger samples will produce a thick white layer between the aqueous and organic phase. This fraction contains DNA. If this layer can be removed without disturbing the other phases, do so and put it in a separate tube (tube B2).</li>
	<li>Isolate RNA as follows.
	<ol>
		<li>From the clear aqueous phase in tube A, precipitate the RNA with 500 &#181;L of 100% isopropanol. Incubate for 10 min at RT. Then, centrifuge tube A at 13,500 x g for 10 min at 4 &#176;C. 
		NOTE: A small white pellet of RNA should be visible at the bottom of the tube.</li>
		<li>Decant the majority of the supernatant. Remove the rest with a 1 mL syringe with a needle and discard the supernatant. 
		NOTE: The size of the needle is not important; however, a larger needle gauge will offer more control so as not to disturb the pellet. A micropipette can be used if needles are not available.</li>
		<li>Add 1 mL of 75% ethanol to tube A to wash the pellet. Spin the tube at 5,300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant from the pellet by decanting and using a 1 mL syringe with a needle, as described in step 2.5.2, and discard it.</li>
		<li>Let the pellet air-dry for 5&#8211;10 min, but do not let it overdry. Use 50 &#181;L of RNase-free water to reconstitute the pellet of RNA before it becomes completely transparent. 
		NOTE: This is an adequate starting volume for 1,000&#8211;3,000 worms, but it may need to be adjusted depending on how much starting material was used.</li>
		<li>Incubate the pellet at 55&#8211;60 &#176;C for 10 min to dissolve it.</li>
		<li>Measure the concentration and the purity using a spectrophotometer. Record absorbances at 260 nm for RNA concentration and at 230 and 280 nm to identify any impurities.</li>
		<li>Clean the RNA to remove any contaminants, such as phenol residue or DNA, either with column purification or with subsequent ethanol washes and DNase treatment. 
		NOTE: At this point, the RNA is ready to be sent for RNAseq analysis or can be used to make cDNA to use for RT-qPCR (see section 3.1 for details). RNA can be stored at -80 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Isolate DNA as follows.
	<ol>
		<li>From the pink organic phase in tube B and the sample in tube B2, if any, precipitate the DNA by adding 300 &#181;L of 100% ethanol and mix by inversion. Leave the tube(s) at RT for 2&#8211;3 min.</li>
		<li>Centrifuge tubes B and B2 at 375 x g for 5 min at 4 &#176;C to pellet DNA.</li>
		<li>Move the supernatant from tubes B and B2 by pouring it into a new 2 mL tube (tube C) and leave it on ice for subsequent protein isolation. Wash the DNA pellet in tube B or B2 with 1 mL of 0.1 M sodium citrate in 10% ethanol for 30 min. Centrifuge tubes B and B2 at 375 x g for 5 min at 4 &#176;C. 
		NOTE: Sodium binds to the DNA backbone, making DNA precipitate more readily in sodium citrate and ethanol, allowing impurities to be removed by low-speed centrifugation.</li>
		<li>Repeat the wash step as described in step 2.6.3. Resuspend the pellet in 1.5 mL of 75% ethanol and leave it at RT for 20 min, with occasional mixing by upending.</li>
		<li>Centrifuge tube B and B2 at 375 x g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant and allow it to dry for 5&#8211;10 min.</li>
		<li>Dissolve the pellet in 150 &#181;L of 8 mM sodium hydroxide. Adjust to the desired pH with HEPES if necessary. Spin the sample at 375 x g for 5 min at 4 &#176;C.</li>
		<li>Using a micropipette, move the supernatant (DNA) to a new tube. Measure the concentration and determine the purity using a spectrophotometer. Record absorbances at 260 nm for DNA concentration and at 230 and 280 nm to identify any impurities.</li>
	</ol>
	</li>
	<li>Isolate protein as follows.
	<ol>
		<li>To precipitate the protein, add up to 1.5 mL of 100% isopropanol to the pink supernatant in tube C, mix by inverting several times, and incubate at RT for 10 min.</li>
		<li>Centrifuge tube C at 13,500 x g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernatant and wash the pellet with 2 mL of 0.3 M guanidine hydrochloride in 95% ethanol for 20 min at RT. Centrifuge tube C at 5,300 x g for 5 min at 4 &#176;C.</li>
		<li>Repeat the wash step as described in step 2.7.3 2x.</li>
		<li>Move the protein pellet to a new 1.5 mL tube (tube C2) and add up to 1.5 mL of 95% ethanol. Vortex and let it sit at RT for 20 min.</li>
		<li>Centrifuge tube C2 at 5,300 x g for 5 min at 4 &#176;C. Discard the supernatant and let the pellet dry for 10 min at RT. Dissolve the pellet in 300 &#181;L of 5% SDS at 50 &#176;C for 60 min. 
		NOTE: A longer incubation time may be required to fully dissolve the protein pellet. In the past, the pellet has been incubated for up to 6 h without sacrificing quality.</li>
		<li>Centrifuge tube C2 at 13,500 x g for 10 min at 17 &#176;C. Move the supernatant to a new tube.</li>
		<li>Measure the concentration using a preferred protein quantification assay that is compatible with detergent. 
		NOTE: The protein is ready to be used in SDS-PAGE and western blotting. It can be stored at -20 &#176;C for future use. For other techniques, such as LC-MS/MS, the detergent will need to be dialyzed off.</li>
	</ol>
	</li>
</ol>
3. Evaluating mRNA and Protein Levels
<ol>
	<li>RT-qPCR
	<ol>
		<li>Prepare cDNA by reverse transcription with 1 ng of RNA, using the following thermocycler protocol: priming (5 min at 25 &#176;C), reverse transcription (20 min at 46 &#176;C), and RT inactivation (1 min at 95 &#176;C).</li>
		<li>Make a stock plate of cDNA by adding 100 &#181;L of 1:100 dilutions of each cDNA sample to individual wells of a 96-well plate. Include appropriate controls, such as no template/water-only wells, and serially diluted samples from 1:25 to 1:400 (originated with pooled cDNA from all of the samples analyzed) to allow for a standard curve to establish primer efficiency for each gene analyzed. 
		NOTE: This format can be used with the 96-well microdispenser, which allows for the transfer of all samples in a plate to an RT-qPCR plate and for the mitigation of well-to-well pipetting variations. The appropriate master mix (e.g., SYBR + primers) can be added to each well thereafter, with a multichannel pipette. Overall, this approach reduces errors introduced when hand-pipetting each sample, thus further reducing variability.</li>
		<li>Add 3 &#181;L of cDNA from the stock plate to the wells of the RT-qPCR plate.</li>
		<li>Make a master mix for each set of primers that includes 1x supermix containing a DNA-intercalating cyanine dye, 5 &#181;M each of forward and reverse primers, and water up to 7 &#181;L per sample. Add 7 &#181;L to the appropriate wells of an RT-qPCR plate and agitate lightly to mix. 
		NOTE: Include samples to measure housekeeping genes to use as a reference to normalize the results20.</li>
		<li>Run the plate using an RT-qPCR protocol that is suitable for the primers being used.</li>
	</ol>
	</li>
	<li>Western blot 
	NOTE: For detailed information about western blots, please refer to Mahmood and Yang21.
	<ol>
		<li>Prepare samples for SDS-PAGE by combining 20 &#181;g of protein with an equal volume of 2x Laemmli Sample Buffer and boil at 95 &#176;C for 10 min.</li>
		<li>Load the samples onto a 4%&#8211;15% Tris-glycine gel and run them at 150 V for 1 h, or until the dye front reaches the bottom of the gel.</li>
		<li>Transfer proteins from the gel to a nitrocellulose membrane at 25 V for 30 min in a semidry transfer apparatus.</li>
		<li>Confirm the proper transfer by staining the membrane with Ponceau S stain.</li>
		<li>Thoroughly wash the stain from the membrane with Tris-buffered saline (TBS) with 0.01% polysorbate 20 (TBS-T).</li>
		<li>Block the membrane with 5% nonfat milk in TBS-T for 1 h at RT.</li>
		<li>Incubate the membrane in the primary antibody at the appropriate dilution according to the supplier&#8217;s recommendations. Leave it on the rocker at 4 &#176;C overnight.</li>
		<li>Wash the membrane 3x, for 5 min each, with TBS-T.</li>
		<li>Incubate the membrane in the appropriate secondary antibody at the appropriate dilution according to the supplier&#8217;s recommendations. Leave it on the rocker for 1 h at RT.</li>
		<li>Wash the membrane 3x, for 5 min each, with TBS-T.</li>
		<li>Image and quantify the western blot, using preferred methods.</li>
	</ol>
	</li>
</ol>

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R., 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=C.+elegans+S6K+Mutants+Require+a+Creatine-Kinase-like+Effector+for+Lifespan+Extension.">C. elegans S6K Mutants Require a Creatine-Kinase-like Effector for Lifespan Extension.</a> Cell Reports. 14 (9), 2059-2067 (2016).</li><li>Larance, 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=Global+Proteomics+Analysis+of+the+Response+to+Starvation+in+C.+elegans+.">Global Proteomics Analysis of the Response to Starvation in C. elegans .</a> Molecular and Cell Proteomics. 14 (7), 1989-2001 (2015).</li><li>Yuan, Y., 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=Enhanced+energy+metabolism+contributes+to+the+extended+life+span+of+calorie-restricted+Caenorhabditis+elegans.">Enhanced energy metabolism contributes to the extended life span of calorie-restricted Caenorhabditis elegans.</a> Journal of Biological Chemistry. 287 (37), 31414-31426 (2012).</li></ol>

The authors have nothing to disclose.

Methods for biomolecule isolations, such as DNA, RNA, and proteins, are often optimized without technical overlap or combinations. This is particularly disadvantageous when samples are difficult to obtain, which could lead to harvesting samples under the same conditions at different times. Depending on the cellular pathways, replicates collected at different times may generate variation. This manuscript offers a procedure to circumvent this hurdle by enabling the concurrent isolation and purification of each biomolecule from the same sample of worms, reducing variations introduced by different isolation techniques, the timing of sample harvest, or unequal harvesting. Controlling these variables not only saves time and resources, but also facilitates reproducibility. Here, we demonstrate a combinatorial approach that avoids compromising RNA and protein quality, albeit with variable results with DNA. Preparations can be further optimized using DNA cleaning procedures. We demonstrated the approach using material from the nematode C. elegans and HeLa cells.
Previous work exploring the transcriptome and proteome of N2 WT animals and eat-2 and rsks-1 mutants have offered insight into various pathways, including mechanisms that extend lifespan4,34,35,36. In an effort to investigate the mechanism of caloric restriction in extending lifespan, a Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) analysis found that eat-2 worms have an overall downregulation of global protein synthesis 36. The data presented here is consistent with this finding, even as the mRNA levels of the same targets are greatly increased. Another group aimed to identify effectors of S6K-mediated longevity and, thus, performed a proteomic screen of rsks-1 worms34. From the RNAseq data found with the current study, we identified at least three genes that corroborate with proteins identified from this screen; MRP-1 and homologs of CPA and neuroligin (F07C4.12) were discovered as being differentially expressed in rsks-1 worms as compared to WT N234.
The data generated using this method is consistent with previous multiomic investigations. The mRNA levels of nine targets were used to predict the protein levels in each sample. Of these targets, many had predictable protein levels based on the mRNA levels. Nevertheless, there were notable discrepancies between the mRNA and protein levels. Importantly, the protocol presented here allows scientists to confidently evaluate and interpret these differences by removing intersample variability by collecting mRNA and protein from the same sample. Furthermore, we compared the mRNA levels to the protein levels collected from the same sample or collected from a similar but different sample harvested with RIPA. We showed that for a number of the targets, there were much lower levels of protein in the RIPA-extracted sample. Without controlling for the intersample variation, it would be impossible to know if this difference was due to the differential regulation of mRNA and protein.
It is important to keep in mind that there are protocols optimized specifically for these different macromolecules, so if a cross-sectional analysis is not the final goal of the experiment, then it would be pertinent to employ those methods instead. Using GTCp to isolate DNA and protein causes them to become less soluble, requiring the reconstitution of DNA in a weak base, such as NaOH, and solubilizing the protein in a buffer with a high concentration of detergent with heating, at the risk of incomplete solubilization. Additionally, GTCp contains guanidinium thiocyanate and acidic phenol, which inactivates enzymes such as proteases, but will slowly degrade protein over time unless frozen. It will be to the discretion of the researcher to decide if the circumvention of these limitations will be worthwhile.
Importantly, when working with RNA, a sterile technique, keeping the sample on ice unless otherwise noted, and the use of commercially available decontaminating reagent is recommended to keep the RNA intact. Notably, larger samples will need more GTCp to start with, in which each extraction solvent will also need to be scaled up. This protocol does not require any manual homogenization of the worm or cell samples when the correct amount of GTCp is used. In the context of DNA isolation, the yield is highly dependent on the proficiency to recover the organic (pink) layer. Finally, to improve the solubilization of proteins during isolation, increasing the volume of resolubilization buffer or adding other detergents besides SDS may be necessary. Indeed, proteins from an adult-only population of worms instead of a mixture of adults, larvae, and eggs are much easier to resolubilize.
Overall, using this protocol provides an integrated approach to biomolecule isolations and facilitates the interpretation of correlations, or lack thereof, between mRNA and protein levels that can arise from separately harvesting biomolecules from different samples. Using this method can help scientists to identify correctly cases where the translation of mRNA to protein is not correlative and can lead to the deeper investigation of posttranscriptional and posttranslational regulatory mechanisms under various conditions.

Multiomics, the analytical approach that uses a combination of omics, such as genome, proteome, transcriptome, epigenome, microbiome, or lipidome, has become increasingly popular when processing large data sets for disease characterization1,2. Mounting evidence has shown that limiting approaches to a single &#34;ome&#34; provides an incomplete molecular analysis (reviewed by Rotroff and Motsinger-Reif1). Large data sets are generated, particularly when performing screens using high-throughput techniques, but in order to paint a complete picture or to identify the most relevant targets, multiomic approaches are preferable. With the use of multiomics approaches, however, there is the frequent observation of discrepancies between mRNA and protein levels3,4,5,6. Notably, mRNA and protein used for side-by-side transcriptomic and proteomic analyses with RNA sequencing (RNAseq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively, are often obtained from similarly treated samples from different replicates, potentially introducing variation between the same conditions3,4,5,6. Harvald et al. performed an elegant C. elegans starvation time-course study that compared the transcriptome and proteome of wild-type (WT) worms to that of hlh-30 mutant worms that lack an important transcription factor in longevity7. Of note, the RNA and protein were harvested from the same condition replicates, so not from the same sample. Their findings show a low correlation between the mRNA levels and protein levels at each time point (r = 0.559 to 0.628). In fact, their heatmap formed four clusters: Cluster I had a large decrease in mRNA levels but little or no decrease in corresponding protein levels, Cluster II had little or no increase in mRNA levels but an increase in protein levels, Cluster III had an increase in mRNA levels but a decrease in protein levels, and Cluster IV had an increase in mRNA levels but only a subtle change in protein levels4. In addition, this intersample variation may be introduced in cases where the samples of the same condition are not collected at the exact same time. For example, mRNA and proteins regulated by the circadian cycle fluctuate depending on the time of day8,9, or, more specifically, the exposure of C. elegans to light9; expression of these circadian proteins may be delayed up to 8 h after gene expression induction10. Nevertheless, the prevalence of this observation does not necessarily mean it is wrong; in fact, this may prove to be informative. Protein and mRNA are in a constant dynamic state between formation and degradation. Moreover, proteins are often posttranslationally modified to increase stability or to induce their degradation11. For instance, their ubiquitination status can lead to either activation or targeting to the proteasome or lysosome for degradation12. Additionally, noncoding RNAs play an important role in regulating gene expression at the transcriptional and posttranscriptional stages13. Thus, the question is how to limit the variables to confirm that the discrepancies we observe in these nematode studies are real.
Here, we propose a method that removes the intersample variable by allowing analyses of different macromolecules from the same sample. The goal of this protocol is to offer a method to consistently isolate DNA, RNA, and protein from a single sample of C. elegans (also referred to as worms) in an effort to reduce variation, improve reproducibility, and facilitate interpretations. Additional benefits of using the same sample include the saving of time and resources during sample collection, facilitating the cross-sectional analysis of valuable and limited samples, including strains that are difficult to grow and maintain, and gaining insights into the differential regulation of macromolecules based on intrasample variations in mRNA and protein levels.
This method is suitable for assessing gene expressions and protein levels from a single sample of worms, allowing for a more comprehensive evaluation of multiple levels of molecular processing. Guanidinium thiocyanate-phenol-chloroform (GTCp) reagent14, a commonly used chemical to isolate RNA, is used for the extraction of nucleic acids and protein from the worms, with subsequent precipitation, washing, and solubilization of each. This protocol is a compilation of various protocols15,16&#160;with minor modifications, designed with a focus on C. elegans, but we have also successfully isolated RNA, protein, and DNA from a pellet of HeLa cells following the same steps. Although not tested here, this protocol is likely to work on tissues as well17,18.

<table>
	<tbody>
		<tr>
			<td>4&#8211;15% Mini-PROTEAN&#174; TGX Stain-Free&#8482; Protein Gels</td>
			<td>BIORAD</td>
			<td>4568084</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CePAS7-s</td>
			<td>DHSB- U of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CeTAC1-s</td>
			<td>DHSB- U of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DLG1-s</td>
			<td>DHSB- U of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GRP78</td>
			<td>Novus Bio.</td>
			<td>NBp1-06274</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat Anti-Mouse</td>
			<td>Li-Cor</td>
			<td>926-80010</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat Anti-Rabbit</td>
			<td>Li-Cor</td>
			<td>92680011</td>
			<td></td>
		</tr>
		<tr>
			<td>HSP60-s</td>
			<td>DHSB- U of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LMP1-s</td>
			<td>DHSB- U of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRP-1</td>
			<td>Abcam</td>
			<td>ab24102</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroligin 3</td>
			<td>Abcam</td>
			<td>ab172798</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Millipore</td>
			<td>MAB1501R</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imaging System</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>C298-500</td>
			<td>Health hazard, Irritant, Toxic</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue</td>
			<td>ThermoSci</td>
			<td>20279</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein assay</td>
			<td>BIORAD</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoch 2 microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (200 proof)</td>
			<td>Fisher</td>
			<td>04-355-223</td>
			<td>Flammable, health hazard</td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>BSL2</td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix</td>
			<td>BIORAD</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydra microdispenser: Matrix Hydra</td>
			<td>Robbins/ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>A516-4</td>
			<td>Flammable, health hazard</td>
		</tr>
		<tr>
			<td>M9 buffer: 
			3 g KH2PO4 
			6 g Na2HPO4 
			5 g NaCl 
			Add H2O to 1 liter. Sterilize by autoclaving. 
			After solution cools down, add 1 mL sterile 1 M MgSO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Sample Buffer (2x)</td>
			<td>BIORAD</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>ThermoSci</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE apparatus</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Alfa Aesar</td>
			<td>J60744</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>IDT</td>
			<td></td>
			<td>25 nmole DNA Oligo with Standard Desalting</td>
		</tr>
		<tr>
			<td>dlg-1: F (5&#39;-GGTCCTACCA GGCAGTTGAG-3&#39;) 
			 R (5&#39;-CACGTCCGTT AACCTCTCCC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hsp-4: F (5&#39;-AGAGGGCTTT GTCAACCCAG-3&#39;) 
			 R (5&#39;-TCGTCAGGGT TGATTCCACG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hsp-70: F (5&#39;-CGGCATGTGA ACGTGCTAAG-3&#39;) 
			 R (5&#39;-GAGCAGTTGA GGTCCTTCCC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hsp-60: F (5&#39;-ATTGAGCAAT CGACGAGCGA-3&#39;) 
			 R (5&#39;-CAACACCTCC TCCTGGAACG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lmp-1: F (5&#39;-ACAACAACAC CGGACTCACG-3&#39;) 
			 R (5&#39;-ATCGAGCTCC CACTCTTTGG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tac-1: F (5&#39;-AGTGGCAGGC AAAGTTCCTC-3&#39;) 
			 R (5&#39;-TGAGCACCTT GATCTCGTCG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pas-7: F (5&#39;-GTACGCTCAA AAGGCTGTCG-3&#39;) 
			 R (5&#39;-CTGAATCGGC ATTGGCTCAC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mrp-1: F (5&#39;-TTTGCCTTGC GCTTGTTCTG-3&#39;) 
			 R (5&#39;-AGTTCCAGTG CGGAGCATAC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F07C4.12: F (5&#39;-TGCTGAGCAT GAAGGACTGT-3&#39;) 
			 R (5&#39;-TGGCAATAGC TCCTCCGTTG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK Actin: F (5&#39;-CTACGAACTT CCTGACGGACAAG-3&#39;) 
			 R (5&#39;-CCGGCGGACT CCATACC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK cyn-1: F (5&#39;-GTGTCACCAT GGAGTTGTTC-3&#39;) 
			 R (5&#39;-TCCGTAGATT GATTCACCAC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK nhr-23: F (5&#39;-CAGAAACACT GAAGAACGCG-3&#39;) 
			 R (5&#39;-CGATCTGCAG TGAATAGCTC-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK ama-1: F (5&#39;-TGGAACTCTG GAGTCACACC-3&#39;) 
			 R (5&#39;-CATCCTCCTT CATTGAACGG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK cdc-42: F (5&#39;-CTGCTGGACA GGAAGATTACG-3&#39;) 
			 R (5&#39;-CTCGGACATT CTCGAATGAAG-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HK pmp-3: F (5&#39;-GTTCCCGTGT TCATCACTCAT-3&#39;) 
			 R (5&#39;-ACACCGTCGA GAAGCTGTAGA-3&#39;)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 qPCR machine</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer: 10 mM Tris-Cl (pH 8.0) 
			1 mM EDTA 
			1% Triton X-100 
			0.2% SDS 
			140 mM NaCl 
			1 tablet of Roche protease inhibitor per 20 ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Supermix</td>
			<td>BIORAD</td>
			<td>1725274</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Max Sens Substrate</td>
			<td>ThermoSci</td>
			<td>34095</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Transfer apparatus</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer Pack</td>
			<td>BIORAD</td>
			<td>170-4159</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>Health hazard (skin, eyes)</td>
		</tr>
		<tr>
			<td>Worm strains:</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2 (wild type)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>eat-2 (MAH95)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rsks-1 (VB633)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined nucleotide and protein extractions in caenorhabditis elegans

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

The RNA, DNA, and protein samples were analyzed, and representative results using the RNA and protein isolates are presented here. Additionally, we compare the protein sample harvested using the GTCp method to the common RIPA lysis method22. For our experiment, we used four independent replicates of each WT (N2) worms and two long-lived mutant worms, eat-2 and rsks-123,24.
RNA and DNA quantity and quality
The concentration of RNA when resuspended in 50 &#181;L of water ranged from 0.2&#8211;2 &#181;g/&#181;L, using around 3,000 adult worms as starting material. The absorbance ratios, which indicate purity, ranged from 2.0 to 2.2 for A260/A280 and 1.9 to 2.5 for A260/A230 (Table 1), indicating the successful extraction of RNA without contamination with GTCp components, such as phenol or guanidine hydrochloride.
The DNA extraction was successful, but the quality was variable. The DNA concentration when resuspended in 150 &#181;L of NaOH ranged from 0.04 to 1.1 &#181;g/&#181;L. The absorbance ratios ranged from 1.8 to 2.1 for A260/A280 and 0.9 to 1.6 for A260/A230 (Table 1), indicating the successful extraction of DNA; however, some of the samples were contaminated with GTCp components, like phenol or guanidine hydrochloride. If DNA isolation is necessary, care must be taken when separating the middle layer from the GTCp phase separation, and more washes may be necessary.
RT-qPCR of selected targets
The RNA isolation from four independent samples of each worm strain was successful and was sent for RNAseq analysis, with subsequent RT-qPCR performed using a supermix containing a cyanine dye. Targets analyzed by RT-qPCR were selected from a large RNAseq dataset based on the most differentially regulated genes common in both mutants as compared to the WT worms. F07C4.1225, a homolog to human neuroligin 3, isoform b, was the selected shared upregulated target. We also included a differentially regulated gene between both mutants, mrp-126, for additional analysis, which was upregulated in eat-2 worms but downregulated in rsks-1 worms. Expression changes of mrp-1 were confirmed via RT-qPCR; however, the F07C4.12 upregulation in rsks-1 worms predicted by the RNAseq analysis was not seen by RT-qPCR (Figure 1).
Additional targets with antibodies validated for worms were investigated as well. These included several organelle markers characterized in Hadwiger et al.27. A number of these markers were differentially expressed at the mRNA level in the mutant worms. As seen in Figure 1, lmp-128&#160;and dlg-129&#160;were upregulated in eat-2 worms; hsp-430, hsp-7030, and lmp-1 were upregulated in rsks-1 worms; hsp-6031&#160;and pas-732&#160;were downregulated in eat-2 worms; hsp-60 and tac-133&#160;were downregulated in rsks-1 worms.
Simultaneous vs. RIPA protein preparation
To affirm the efficiency and quality of the protein with the GTCp method as described above, we compared it to protein collected using the more traditional method of RIPA lysis22. Using the same amount of starting material, namely either adults only (about 10,000 worms) or a mixed population of adults, larvae, and eggs (about 130,000 worms), the overall quantity collected with GTCp was less when resuspended in equal end volumes (Table 1); however, the extraction from an adult-only population was more successful than from a mixed population. Additionally, the quality of the proteins was similar, albeit the GTCp-extracted protein showed a better resolution of larger proteins upon equal loading of total protein, as shown by Coomassie stain in Figure 2. Indeed, targets evaluated by western blot in Figure 3 showed similar levels in most cases; however, the proteins larger than 75 kDa showed lower levels in the RIPA-extracted protein compared to the GTCp-extracted protein (Figure 3A, right lane; Figure 3B, yellow bar).
The extraction method presented here was also evaluated in the mammalian cell line HeLa. For reference, the amount of protein extracted with RIPA from a compact pellet of six million HeLa cells (an ~25 &#181;L pellet) was comparable to that extracted from a 130,000 mixed population of worms (an ~75 &#181;L compact pellet), or about half of what is extracted from 10,000 adult worms (an ~100 &#181;L gravity-settled pellet), as seen in Table 1. We show that the efficiency of protein extraction (Table 1) and the resolution of larger proteins (Figure 2) was decreased in GTCp compared to RIPA-extracted protein in HeLa cells, suggesting this technique works better in an adult population of worms. The incomplete solubilization of the protein pellet from GTCp-extracted HeLa protein may be a limitation of this method in mammalian cells.
Immunoblotting targets analyzed by RT-qPCR
Next, we investigated the protein levels of the gene products tested by RT-qPCR to determine if the mRNA levels correlated with the protein levels. As seen in Figure 3, the average expression changes of protein correlated with the average mRNA expressions changed for many targets; however, some protein levels did not reflect the changes in mRNA levels. Often, the mRNA levels in eat-2 worms were higher than those in the other strains, but the protein levels were either the same or lower than the other strains of the same target. The most outstanding observation was the very high levels of dlg-1 mRNA in eat-2 worms, which did not translate to more protein; in fact, there were lower dlg-1 protein levels when compared to the other strains (Figure 3).
Finally, the levels in the GTCp-extracted protein and the RIPA-extracted protein showed a striking difference. The RNA was extracted in the same manner for both; however, the similar but separate sample collected by RIPA lysis showed markedly reduced protein levels, particularly for those larger than 75 kDa (Figure 3A, right lane; Figure 3B, yellow bar).
Intrasample comparison of mRNA and protein levels
One of the goals of this protocol was to determine if the variation we saw at the mRNA and protein level was real, or if it could be an artifact of intersample variation. In Figure 4, a subset of targets was compared within the single samples. Each individual sample is shown as the same shade and color of the dot, with sample 1 being the darkest shade and sample 4 as the lightest shade of gray (WT), blue (eat-2), or red (rsks-1). Within most samples, there was a low variability of the mRNA levels, with greater variability at the protein level, a potential flaw of the semiquantitative analysis of western blots. When looking at the position of each of the colored dots between the mRNA and protein pairs, the order of highest-to-lowest often did not match; for example, in WT, the hsp-60 mRNA order was sample 4, 3, 2, 1, but the protein levels were 1, 2, 3, 4. Thus, differences between mRNA and protein levels within a sample certainly exist, but the presented method allows users to remove the time of collection as a possible source of the observed difference.
<img alt="Table 1" src="/files/ftp_upload/59178/59178table1.jpg" /> 
Table 1: Concentrations and absorption ratios. RNA and DNA concentrations and purity ratios were measured on a spectrophotometer. Protein concentrations were determined using a colorimetric protein quantification assay. The gray, blue, and red highlight the samples used for RT-qPCR and western blot. The yellow indicates samples used to compare GTCp to RIPA protein extraction or worm protein to HeLa protein. <a href="https://www.jove.com/files/ftp_upload/59178/Table_1.xlsx">Please click here to download this file.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59178/59178fig1.jpg" /> 
Figure 1: Gene expression by RT-qPCR. RT-qPCR analysis for the mRNA obtained by this method, confirming targets identified from RNAseq data, and of organelle marker genes. The error bars represent standard deviation; n = 4. The concentration of mRNA was defined against a standard curve for each set of primers. All mRNA levels were normalized to the average of a set of six housekeeping genes used as reference genes, which include act-1, cdc-42, ama-1, nhr-23, pmp-3, and cyn-1. <a href="https://www.jove.com/files/ftp_upload/59178/59178fig1large.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/59178/59178fig2.jpg" /> 
Figure 2: Comparison of GTCp- versus RIPA-extracted protein in worms and HeLa cells. Total protein from wild-type (WT) worms or HeLa cells harvested via GTCp or RIPA lysis method, separated by SDS-PAGE, and stained with Coomassie blue. Each lane contains 25 &#181;g of total protein. <a href="https://www.jove.com/files/ftp_upload/59178/59178fig2large.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/59178/59178fig3.jpg" /> 
Figure 3: Protein levels in worms. (A) Western blot of targets investigated by RT-qPCR and (B) densitometry analysis of signal intensity. Each lane contains 20 &#181;g of total GTCp-extracted protein from wild-type (WT), eat-2, or rsks-1 mutant worms. The image shown is a representation of four independent replicates per worm strain. &#946;-actin (bottom) was used to control for equal loading for each target; only one representative set is shown. The right lane contains 20 &#181;g of total RIPA-extracted protein from rsks-1 mutant worms. (B) Corresponding signal intensities were quantified using ImageJ. The error bars represent standard deviation; n = 4. <a href="https://www.jove.com/files/ftp_upload/59178/59178fig3large.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/59178/59178fig4.jpg" /> 
Figure 4: Intrasample mRNA and protein level comparison. The mRNA and protein levels from individual samples from a subset of targets for each strain are aligned. The color is the representative of the Table 1. The gray/black is WT, the blue is eat-2, and the red is rsks-1. mRNA and protein of the same target are paired next to each other and separated by nematode strain. <a href="https://www.jove.com/files/ftp_upload/59178/59178fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Combined Nucleotide and Protein Extractions in Caenorhabditis elegans at JoVE.com

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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

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10.1K Views.  Brown University. Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Video: Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans