The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.
Comprised of only 20 cells, the Caenorhabditis elegans intestine is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.
The Caenorhabditis elegans nematode worm, with a mere 959 cells and a 4 day egg-to-egg life cycle, is an ideal model system for many genetics, genomics, and developmental studies1,2. The ease of forward and reverse genetic screening, the prevalence of engineered fluorescent markers, the capacity to perform nucleotide-specific genome editing, and the numerous community-wide resources have all contributed to major discoveries and insights in the C. elegans system. However, a significant drawback is the difficulty of obtaining pure populations of cells, tissues, or organs, which are small, fragile, and can be interconnected. As pure populations of cells are important for genomics assays such as RNA-seq, ChIP-seq, and ATAC-seq, several approaches have emerged to obtain pure preparations of C. elegans cells, tissues, and organs. Here, a method for hand dissecting intestines, in large sections, out of adult C. elegans worms is described. The resulting preparations are suitable for downstream genomics assays (Figure 1).
The fine-scale tissue dissection method described here (Figure 2) is just one approach. Other alternative techniques-such as molecular tagging, disaggregating worms, and purifying cell types of interest with fluorescence-activated cell sorting (FACS) and post hoc analysis-have also been successfully used to survey the tissue-specific features of C. elegans molecular biology. An advantage of hand dissection over these other approaches, however, is that it can be used to simultaneously explore the features of the C. elegans intestine and its bacterial contents3,4,5. This enables 16S rRNA gene sequencing and facilitates microbiome studies within the C. elegans system. An important limitation, however, is that intestinal cells are not individually isolated.
Molecular tagging imparts a cell type-specific tag to molecules only within the specified tissue or cells of interest. These tags can then be isolated from total worm preparations. In this way, tissue-specific promoters driving a tagged polyA-binding protein or spliced leader have enabled tissue-specific transcriptome profiling6,7,8,9,10 and 3'UTR mapping11,12. Similarly, tissue-specific transcription factor profiles have been conducted using ChIP-seq and DamID, in which promoter-specific transcription factor variants were appended with tags or enzyme fusions13,14.
FACS allows for isolating cell types of interest from dissociated worms based on their intrinsic cellular characteristics and fluorescent properties. This approach has generated tissue-specific transcriptomes from diverse organs8,15,16 and individual neuronal cell types8,9,15,16,17,18 and has been used to create an expression map of the entire C. elegans nervous system19,20. FACS, and its cousin fluorescence-activated nuclei sorting (FANS), have also been used to generate cell-specific chromatin profiles21,22.
Finally, post-hoc analysis can be performed in single-cell resolution assays. In this method, all individual cells are surveyed, the cell type of each is ascribed in the analysis stage, and the cell types of interest are selectively filtered for further study. Post-hoc analysis has been successfully used to obtain transcriptomes of developing cells with both high spatial and temporal resolution in C. elegans embryos23,24,25,26,27 and L128 stage worms. Chromatin accessibility has also been characterized using ATAC-seq instead of RNA-seq using a similar strategy29.
Each approach has its advantages and limitations. For the C. elegans intestine, worm disaggregation and FACS isolation of intestinal cells is achievable in the embryo and larval stages30 but is challenging in adults. This is thought to be due to the intestine's large, endo-reduplicated, and strongly adherent cells making them difficult to dissociate undamaged. The hand dissection method described here circumvents these challenges, allowing for the isolation of large sections of the adult worm's intestine. The practice of hand dissecting gonads from this same stage is widespread and straightforward. Intestine dissection is similar to gonad dissection but less commonly performed32. The protocol presented here is adapted from a longer, unpublished protocol developed by Dr. James McGhee and Barb Goszczynski. This streamlined protocol borrows techniques for isolating blastomeres from early-stage embryos23,33,34,35. Though hand dissection is not feasible for isolating most cell or tissue types in C. elegans, it is ideal for isolating intestines from adult worms. Therefore, hand dissection complements other means for obtaining intestine-specific cell preparations.
CL2122 worms were used for the present study. The worms were obtained through the Caenorhabditis Genetics Center (CGC, see Table of Materials), funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
1. Growing of worms for dissection
2. Preparation of stock solutions and microcapillary pipettes
NOTE: Table 1 provides the details of all the buffers and solutions used for the present study.
3. Experimental preparation
4. Hand dissection of the C. elegans intestine
5. RNA isolation from dissected intestines
The present protocol was used to isolate large sections of the intestine from adult C. elegans by hand (Figure 2). The final intestine sample for each experimental group shown is comprised of an equal collection of anterior-mid and mid-posterior intestine sections. However, depending on the experimental question, it could also comprise a collection of only anterior, mid, or posterior intestine sections. Collectively, three representative results are presented for this protocol. The first depicts the successful dissection and isolation of intestines (Figure 6). The second reports the results of RNA isolation from isolated intestines (Figure 7). The third shows the results of microbial surveillance from isolated intestines (Figure 8).
For the first result, Figure 6A displays what an extruded intestine looks like after making a primary incision at site "a" in Figure 5A within adult CL2122 worms. As CL2122 worms harbor the intestine-specific mtl-2 promoter fused to GFP (mtl-2p::GFP), isolated intestines will glow green under the fluorescent dissecting scope. The successful dissection of an intestinal segment is then shown in Figure 6B. This intestine section is free from visible contaminants such as debris from the gonad or carcass. In contrast, Figure 6C displays intestines that are not successfully dissected, as gonad and carcass are still visibly attached to the intestinal segment.
For the second result, the intestines were harvested into a nucleic acid isolation reagent and processed the next day using a low-input total RNA extraction protocol (see Table of Materials). A final intestine sample of 60 total intestine sections from the anterior-mid and mid-posterior sections yields roughly 15 ng of high-quality total RNA (Figure 7A). This amount of total intestines can easily be obtained in a single day but can also be broken up over several days if needed. RNA yields from hand dissection are more efficient than worm disaggregation and FACS isolation of intestinal cells in that hundreds of thousands of intestinal cells are required to obtain commensurate quantities of total RNA (Figure 7B). Importantly, the RNA yields generated by hand dissection are more than sufficient as input for commercial RNA-seq library kits (i.e., NEBNext Ultra II and NEBNext Single Cell/Low Input), which can take as little as 2 pg of RNA.
For the third result, the intestines were harvested into sterile ddH20 and processed the same day using a commercial microbial DNA isolation kit (see Table of Materials). A final intestine sample of 40 total intestine sections from the anterior-mid and mid-posterior sections yields around 0.009 pg of total microbial DNA using a pan-bacterial detection assay (see Table of Materials) (Figure 8). These quantities are too low for traditional quantification methods and must be extrapolated from qPCR standard curves. Ideally, users should target a final total amount of intestines >40 as this increases the detection limit and boundary of signal-to-noise regarding reagent contamination levels.
When considering experimental design, collecting appropriate control samples is always necessary. For transcriptomics experiments, a suitable experimental control can include preparations of the whole worm collected in the same manner as intestines. During the dissection and isolation of intestines from C. elegans, however, it is common to see bits of carcass and gonad clinging to the intestine sections. While ideally these contaminating tissues will be removed from the intestines prior to storage for downstream use, additional experimental controls can include the collection of the leftover worm carcasses post intestine dissection and/or the collection of dissected gonads. For microbiome experiments, controls can also include preparations of the whole worm collected in the same manner as intestines, in addition to conventional positive (bacterial culture) and negative (water) controls.
Figure 1: Hand dissection of intestines from adult C. elegans used to generate tissue-specific preparations for use in a wide variety of downstream omics assays. Please click here to view a larger version of this figure.
Figure 2: Scheme for the hand dissection protocol. This protocol was used to isolate large sections of the intestine from adult C. elegans by hand. Intestines can be isolated for different downstream assays. Shown here is the use of intestines for RNA isolation and microbial DNA isolation. Image created with BioRender.com. Please click here to view a larger version of this figure.
Figure 3: Microcapillary pipette fabrication. (A) A freshly pulled but unforged microcapillary pipette is shown through the ocular piece of a microforge. An ocular ruler is used to measure 50 µm sized microcapillary pipettes to an estimated inner diameter of three tick marks (shown, indicated by arrow). (B) Unforged, 50 µm and 100 µm microcapillary pipettes are shown under the dissecting scope alongside a calibration ruler with 1 div = 0.1 mm. (C) The 50 µm and 100 µm microcapillary pipettes from (B) are shown again but from a different vantage point. Please click here to view a larger version of this figure.
Figure 4: The use of chelation buffer (CB) during hand dissection to improve RNA yield. A low input total RNA extraction protocol generated RNA preparations from named tissues. Representative gel electrophoresis runs characterizing isolated total RNA quality (RIN, RNA integrity number) and quantity are shown. Please click here to view a larger version of this figure.
Figure 5: Hand dissection steps. (A) The dissection steps described in the protocol are outlined here. In (1), the intestine of the worm is visualized by GFP fluorescence. The primary incision site can be evenly distributed among the worms to ensure even coverage across the intestine's entire length. Alternatively, either "a" or "b" primary incision sites can be selected to obtain an anterior-mid or mid-posterior intestinal fragment-specific preparation. In (2), the intestine has extruded, taking on a loop shape. In (3), the intestine is first cut away from the worm body while attempting to liberate it from the carcass and gonad. The intestine is then removed from any remaining carcass or gonad that cannot be removed. In (4), a cleaned section of the intestine is isolated and ready for storage. (B) A critical step is to dislodge any clinging debris (i.e., gonad, carcass) from the intestine by passing it in and out of the microcapillary pipette fashioned at the end of the mouth aspirator. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 6: Representative results of hand dissection steps. A representative image showing (A) the extrusion of the intestine from the worm. These are worms of the CL2122 genotype, expressing GFP under the intestinal cell-specific mtl-2 promoter. Two representative images show (B) a cleaned and fully isolated section of the intestine and (C) an isolated intestine with contaminating gonad and carcass tissues. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 7: Representative results of total RNA extraction from isolated intestines. (A) A representative image of RNA preparations resolved by agarose gel electrophoresis is shown, characterizing the quality (RINs, RNA integrity Number) and quantity of isolated total RNAs. (B) A representative gel of total RNA isolated from intestinal cells harvested from L1 stage worms via fluorescence-activated cell sorting (FACS) is shown for comparison. At 20 cells per intestine, it can be inferred that the hand dissection method yields more total RNA per intestine than FACS-purified intestinal cells. Please click here to view a larger version of this figure.
Figure 8: Representative results for total microbial DNA extraction from isolated intestines. A commercial microbial DNA isolation kit generated DNA preparations from intestines, whole worms, and controls. A pan-bacterial gene assay was used to quantify the number of bacteria in the samples. A representative image of (A) the qPCR amplification curves generated, (B) the E. coli OP50 standard curve used to quantify the samples, and (C) the samples are shown. In (B), the log starting concentrations of E. coli standards are graphed against their CT values. In (C), two replicates (reps) of 40 whole worms were cut medially, and 40 isolated intestinal sections were processed for microbial gDNA isolation. The No-Template-Control (NTC) from the qPCR run is also shown. The Y-axis represents the sample CT values. The amount of DNA quantified from PCRs is shown as total picograms (pg) values above the sample bars. Please click here to view a larger version of this figure.
Table 1: Compositions of the buffers and solutions used in this study. Please click here to download this Table.
This article describes the step-by-step protocol for hand dissecting intestines from adult C. elegans, generating pure preparations for downstream assays. Critical steps in this protocol include (1) ensuring not to over-paralyze the worms, (2) making accurate dissection cuts, (3) forging appropriately sized micro-pipettes for dissection, and (4) ensuring the speedy recovery of healthy intestines during the final harvest. For these reasons, care must be taken when exposing worms to the levamisole solution, and the hypodermic needles need to be refreshed frequently to ensure maximum sharpness. Handling the intestine using the microcapillary pipette and mouth aspirator is another step that will take practice. Properly forged micro-pipettes of the appropriate size also make a substantial difference in isolating large sections of the intestine during dissections, in addition to reducing the risk of losing intestines within the micro-pipette. New protocol users commonly lose intestines on the inner edge of the microcapillary pipette before they can be ejected into the isolation reagent. This problem can be amended with practice and properly forged microcapillary pipettes.
The protocol described herein was designed for use in adult worms. Preliminary trials support that this protocol is also effective for use in L4 worms and older adult worms. However, the efficacy of this protocol has not yet been evaluated in early larval stage worms. A limitation of this approach is the small amount of material it yields. Though the quantities are sufficient for RNA-seq and PCR, they may not be adequate for other assays. As such, users need to determine if the minimum required input for an assay can be feasibly collected with this protocol.
Our lab routinely utilizes FACS to purify intestinal cells post isolation30, post-hoc analysis methods for intestinal cell identification, and this hand dissection method30,42. Hand dissection has the advantage of being amenable for use in adult worms when worm disaggregation and cell isolation are less successful. Furthermore, the efficiency and quality of total RNA extracted from hand dissection preparations are high, likely because the tissues are rapidly plucked from the worms and then quickly deposited in a nucleic acid isolation reagent, reducing RNA degradation. Another benefit of the hand dissection method is that it is low cost, easy to learn, and does not require specialized equipment. Finally, this approach allows for the harvest and isolation of gut bacteria from worm intestines, enabling downstream microbiome studies.
The hand dissection protocol described here for isolating intestines from adult C. elegans represents a powerful tool for studying various aspects of C. elegans biology. For example, with a pure preparation of intestines, researchers can investigate the intersection between immunity, aging, metabolism, and the microbiome.
The authors have nothing to disclose.
We are indebted to the pioneering work of James McGhee and Barb Goszczynski, who initially developed the intestine dissection method from which this protocol is adapted. Our work is supported by a MIRA (R35) Award overseen by the National Institute of General Medical Sciences (National Institutes of Health, R35GM124877 to EON) and an NSF-CAREER Award overseen by the NSF MCB Div Of Molecular and Cellular Bioscience (Award #2143849 to EON).
Acetylated Bovine Serum Albumin (BSA) | VWR | 97061-420 | Nuclease free BSA |
CL2122 worm strain | CGC (Caenorhabditis Genetics Center) | CL2122 | dvIs15 [(pPD30.38) unc-54(vector) + (pCL26) mtl-2::GFP]. Control strain for CL2120. Phenotype apparently WT. |
Calcium Chloride Dihydrate | Fisher | C79 | needed for making Egg Salts |
50 mL Centrifuge Tubes, Bulk | Olympus Plastics | 28-108 | Nuclease free conical tube needed for solution making. |
15 mL Centrifuge Tubes, Bulk | Olympus Plastics | 28-103 | Nuclease free conical tube needed for solution making. |
Concavity slide (2-well) | Electron Microscopy Sciences | 71878-08 | 12-pk of 2-well concavity slides |
Ethylene glycol-bis(2-amino-ethylether)-N,N,N',N'-tetraacetic acid (EGTA) | Millipore Sigma | E3889 | needed for making chelation buffer |
Fluorescent Dissection Microscope | Leica | M205 FCA | This is an optional piece of equipment that can be used with fluorescent C. elegans strains to help guid users during hand dissections |
N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) | Millipore Sigma | H4034 | needed for making dissection buffer |
High Sensitivity RNA ScreenTape | Agilent | 5067-5579 | for assesment of total RNA quality and quantity |
High Sensitivity RNA ScreenTape Ladder | Agilent | 5067-5581 | for assesment of total RNA quality and quantity |
High Sensitivity RNA ScreenTape Sample Buffer | Agilent | 5067-5580 | for assesment of total RNA quality and quantity |
HostZERO Microbial DNA Kit | Zymo Research | D4310 | Isolation of microbial DNA from worm intestines/worms |
Hypodermic Needle (27G x 1/2") | BD Scientific | 305109 | needed for hand dissection of intestines |
Levamisole (a.k.a. (-)-Tetramisole hydrochloride) | Millipore Sigma | L9756 | used to temporarily paralyze worms prior to hand dissection of intestines. |
Luer-Lok General Use Disposable Syringe (1 mL) | BD Scientific | 309628 | Optional. Can be used to affix the hypodermic needle to, allowing easier manipulation of the needle during dissection. Remove the plunger. |
Magnesium Chloride Hexahydrate | Fisher | M33 | needed for making Egg Salts |
Magnesium Sulfate Hpetahydrate | Sigma-Aldrich | 230391-500G | needed for making M9 buffer |
MF-900 Microforge | Narishige | MF-900 | Used to forge the microcapillary pipettes. Available through Tritech Research. |
1.7 mL Microtubes, Clear | Olympus Plastics | 22-282 | Nuclease free microfuge tube needed for solution making and sample storage. |
Mouth Aspirator Tube | Millipore Sigma | A5177 | Mouth aspirator tube is needed in combination with the microcapillary pipette to allow aspiration of dissected intestines. |
16S Pan-Bacterial Control TaqMan Assay | Thermo Fisher | A50137 | Assay ID: Ba04930791_s1. Assay used for gut microbial detection via qPCR. |
P-1000 Micropipette Puller | Sutter Instruments | Model P-1000 | Used to pull the microcapillary pippettes prior to forging. |
Petri Dish (35 x 10 mm) | Genesee Scientific – Olympus Plastics | 32-103 | Used to make M9 bath and Disseciton Buffer bath for washing worms prior to dissection. |
Phenol:Chloroform:IAA | Ambion | AM9730 | Used in the isolation of total RNA |
Potassium Chloride | Millipore Sigma | 529552 | needed for making Egg Salts |
Potassium Phosphate Monobasic | Sigma-Aldrich | P0662-500G | needed for making M9 buffer |
Qubit 3 Fluorometer | Invitrogen | Q33216 | Accompanies the Qubit RNA HS Assay Kit. Can be used to quantify RNA prior to running sample on the Agilent ScreenTape. |
Qubit RNA HS Assay Kit | Invitrogen | Q32852 | Can be used to quantify RNA prior to running sample on the Agilent ScreenTape. |
RNasin Ribonuclease Inhibitor | Promega | N2111 | Broad spectrum inhibition of common eukaryotic Rnases |
RNase-Free DNase Set | Qiagen | 79254 | used for on-column DNA digestion during RNA isolation protocol. |
RNeasy Micro Kit | Qiagen | 74004 | Used for isolation of total RNA from worm intestines/worms |
Standard Glass Capillaries | World Precision Instruments | 1B100F-4 | 4 in OD 1.2 mm standard borosilicate glass capillaries used to make microcapillary pipettes for dissection |
Sodium Chloride | Fisher | S271 | needed for making Egg Salts |
Sodium phosphate dibasic heptahydrate | Fisher Scientific | S373-500 | needed for making M9 buffer |
Syringe filter (0.2 micrometer SCFA) | Thermo Fisher | 72302520 | Optional for use with the mouth aspirator tube when mouth pipetting. |
4150 TapeStation System | Agilent | G2992AA | Accompanies the RNA ScreenTape reagents for assessing RNA quality and quantity |
TaqPath BactoPure Microbial Detection Master Mix | Applied Biosystems | A52699 | master mix used for qPCR |
TRIzol Reagent | Thermo Fisher Scientific | 15596026 | Nucleic acid isolation and preservation. QIAzol (Qiagen; 79306) can be substituted if preferred. |
Worm Pick | NA | NA | Made in house from a pasteur pipette and a platinum wire. See wormbook for details. |