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Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans

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15-Oct 2016



Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm (Lee et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for C. elegans extruded gonads.

Keywords: Active transcription site (活跃转录位点), mRNA (mRNA), smFISH (smFISH), sygl-1 (sygl-1), C. elegans (秀丽隐杆线虫), Gonad (性腺)


smFISH enables direct and precise quantitation of mRNA in vivo. In addition, multiple RNA species can be scored simultaneously in the same cell by multiplexing smFISH probes. There have been previous publications using smFISH in C. elegans, but those studies used wide-field microscopy, which often has lower spatial resolution and requires additional image processing (e.g., image deconvolution) (Ji and van Oudenaarden, 2012). Here we describe an smFISH procedure optimized for the C. elegans germline tissue. Each step is detailed, including use of confocal microscopy to obtain precise measurements. Our protocol minimizes sample-to-sample variability and allows precise quantitation of mRNA and nascent transcripts.

Materials and Reagents

  1. Gloves
  2. RNase-free microcentrifuge tubes (1.5 ml) (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: AM12450 )
  3. Microscope slide (Fisher Scientific, catalog number: 12-544-1 )
  4. High-precision microscope cover glass (Marienfeld-Superior, catalog number: 0107052 ; can be ordered through Azer Scientific)
    Note: Any cover glass compatible with the microscope used for smFISH can be used.
  5. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  6. Aluminum foil
  7. Scalpel (Feather disposable scalpel #10) (Medex Supply, catalog number: GRF-2975#10 ) or needle (25 G) (BD, catalog number: 305125 )
  8. RNase-free filtered tips (Mettler-Toledo, Rainin, catalog numbers: 17007957 , 17002927 and 17014361 for 20, 200 and 1,000 µl tips, respectively)
  9. 0.2 µm syringe filter (EMD Millipore, catalog number: SCGP00525 )
  10. C. elegans. Some strains were provided by the CGC (http://www.cgc.cbs.umn.edu, which is funded by NIH Office of Research Infrastructure Programs [P40 OD010440])
  11. smFISH probe(s) conjugated with fluorophores (LGC BioSearch Technologies, https://www.biosearchtech.com/), see below for probe storage and dilution
  12. RNaseZap® (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  13. ProLong Gold Antifade Reagent Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36930 )
  14. Nail polish
  15. RNase-free 1x PBS (Fisher Scientific, catalog number: BP24384 )
  16. Tween-20 (Fisher Scientific, catalog number: BP337-100 )
  17. Levamisole (or Tetramisole) (Sigma-Aldrich, catalog number: L9756-10G ); store at -20 °C
  18. 37% formaldehyde (AMRESCO, catalog number: 0493-500ML )
  19. Triton X-100 (Fisher Scientific, catalog number: BP151-100 )
  20. Ethanol (Acros Organics, catalog number: 615090010 )
  21. Tris base (Fisher Scientific, catalog number: BP152-5 )
  22. 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9260G )
  23. SSC (20x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9763 )
  24. Formamide, deionized (EMD Millipore, catalog number: 4610-100ML ); store at 4 °C; warm to room temperature prior to opening
  25. DEPC water, sterile (EMD Millipore, catalog number: 9610-1L )
  26. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  27. Dextran sulfate (VWR, catalog number: 97061-196 )
  28. Glucose, nuclease free (Fisher Scientific, catalog number: D16-500 )
  29. Tris, pH 8.0, nuclease free (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9855G )
  30. Glucose oxidase (MP Biomedicals, catalog number: 0 2195196 ); store at 4 °C
  31. Catalase (Fisher Scientific, catalog number: S25239A )
  32. Trolox (Acros Organics, catalog number: 218940050 )
  33. Sodium acetate (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9740 )
  34. Tricaine (Sigma-Aldrich, catalog number: E10521-10G ); store at -20 °C
  35. Vaseline
  36. Lanolin
  37. Paraffin
  38. Fixation: nuclease-free (see Recipes)
    1. 1x PBS + 0.1% Tween-20 (PBSTw)
    2. 1x PBSTw + 0.25 mM levamisole
    3. 1x PBSTw + 3.7% formaldehyde
    4. 1x PBS + 0.1% Triton X-100
    5. 70% ethanol
  39. Probe and hybridization: nuclease-free (see Recipes)
    1. TE buffer
    2. Formamide
    3. smFISH wash buffer
    4. smFISH wash buffer + DAPI
    5. Hybridization buffer (HB)
  40. Reagents for GLOX buffer/mounting medium: nuclease-free (see Recipes)
    1. GLOX buffer, no enzymes
    2. GLOX buffer + enzymes
    3. 10% glucose
    4. Glucose oxidase, 3.7 mg/ml
    5. Catalase
    6. 200 mM Trolox
    7. VALAP


  1. Pipettors (e.g., Mettler-Toledo, Rainin, model: P10, P20, P200, P1000)
  2. Glass Petri dish (e.g., diameter: 5 cm) or depression slide for dissecting worms
  3. Rotator (e.g., BD, Clay Adams Nutator, model: 421105 )
  4. Microcentrifuge at room temperature (Eppendorf, model: 5415 D )
  5. 37 °C incubator
  6. Vortexer (e.g., Baxter, catalog number: S8223-1 )
  7. Confocal microscope (Leica Microsystems, model: Leica TCS SP8 )


Note: Figure 1 shows a flowchart of the protocol. All spins are at 400 x g (2,000 rpm) for 30 sec unless otherwise stated and buffer recipes are listed at the end.

Figure 1. Flowchart of the smFISH protocol for the C. elegans gonad

  1. Probe preparation for use
    smFISH probes can be designed and purchased through the Biosearch Technologies website (https://www.biosearchtech.com/). Choose fluorophores compatible with the microscopic system used for the experiments. For multiplexed probes, carefully choose probe-conjugated fluorophores to eliminate bleed-through between channels during image acquisition. To store smFISH probes, resuspend in 20 µl TE buffer (see Recipes) at pH 8.0 (final concentration of 250 µM) and store at -20 °C. This stock solution can be diluted to an appropriate dilution (e.g., 1:10-1:100) in TE buffer to make the working solution. Choose the dilution that works best for smFISH (e.g., the highest signal-to-noise ratio) and keep diluted probe solution at -20 °C in the dark. The frozen probe solution should be thawed at RT when used for smFISH. In our hands, multiple rounds of freezing-thawing did not affect the performance of smFISH. Do not boil the probes.

  2. Day 1: Gonad dissections for smFISH
    1. Wipe down bench top, gloves, and pipettors with RNaseZap®.
    2. Pick worms from growth plates into glass Petri dish (or depression slide) with PBS with 0.1% Tween-20 (PBSTw) containing anesthesia (e.g., 0.25 mM levamisole [or tetramisole]). Alternatively, add 200-300 µl PBSTw with anesthesia to growth plate and pipet worms into Petri dish or depression slide. A larger volume (1-2 ml) of PBSTw can also be used for moving worms into Petri dish. In case of whole worm staining, skip steps B2-B3 and proceed to step B4.
    3. Extrude gonads as quickly as possible (< 10 min including spin) directly in the Petri dish. Cut behind pharynx or at tail with a #10 scalpel or a needle (Figure 2) (Crittenden et al., 2017).
    4. Pipet extruded gonads into a 1.5 ml microcentrifuge tube. Spin and remove supernatant.
    5. Add 1 ml fix solution (final 3.7% formaldehyde v/v in 1x PBS + 0.1% Tween-20) and incubate for at least 15 min (up to 45 min) at room temperature (RT) on rocker.
    6. Spin down the sample.
    7. Wash 1 x in 1 ml PBSTw.
    8. Incubate for 10 min in 1 ml 1x PBS + 0.1% Triton X-100 at RT to permeabilize.
    9. Wash 2 x with 1 ml PBSTw.
    10. Resuspend in 1 ml 70% EtOH and incubate at 4 °C for 16-18 h (overnight). The sample can be stored in 70% EtOH for up to one week.

      Figure 2. Gonad extrusion. Cut the head (behind the pharynx) or tail of the worm (black dashed lines) using a scalpel or a needle to extrude gonads. Internal pressure of the worm causes gonads to extrude readily. Image: Maria Gallegos.

  3. Day 2: Hybridization
    1. Make wash buffer fresh, letting formamide come to RT before opening.
    2. Bring hybridization buffer (HB) to RT before opening.
    3. Thaw diluted probe, protecting from light.
    4. Spin the extruded gonads prepared on Day 1, remove ethanol.
    5. Add 1 ml wash buffer, equilibrate for 5 min at RT. Spin and remove as much buffer as possible.
    6. Add 1 μl diluted probe to 100 μl HB per sample (final probe concentration of 0.025-0.5 µM) and gently flick the tube (see Day 2 hybridization notes).
    7. Resuspend sample in HB with probe and mix well.
    8. Incubate overnight at 37 °C, with rotation and protected from light.
      Note: You can leave samples for up to 48 h at this step.

  4. Day 3: Wash and mount samples (all at room temperature)
    1. Add 1 ml smFISH wash buffer, invert to mix. Spin and remove buffer.
      Note: If worms don’t pellet well due to HB viscosity, this spin may be done at 400-500 x g (2,200-2,400 rpm) for 1.5 min.
    2. Wash with 1 ml wash buffer + 1 μg/ml DAPI for 30 min on rotator (protected from light).
    3. Wash 2 x with 1 ml wash buffer.
      Note: Either wash 5 min on the rotator or invert tube ~10 times before spinning down again.
    4. Remove as much wash buffer as possible from fixed samples.
    5. Resuspend gonads in the mounting media (e.g., ProLong Gold Antifade Mountant or GLOX buffer).
    6. Let sit for 30 min to a few hours.
    7. Pipet 12-15 µl onto a slide, cover with a 22 x 22 coverslip. Remove excess liquid.
    8. Cure at least overnight and up to 4 days in the dark at room temp or 4 °C. The ProLong Gold user manual recommends a ‘curing’ process (~24 h at RT) for best performance. We have observed that the curing process does improve fluorescence signal.
    9. Seal with nail polish for long term storage.

Data analysis

Representative smFISH image (Figure 3).

Figure 3. sygl-1 smFISH at the distal gonad. The sygl-1 gene is a direct target of Notch transcriptional activation. SYGL-1 protein is a critical regulator for germline stem cell (GSC) maintenance. Individual nascent transcripts (bright white nuclear spots; overlay of yellow and magenta channel, indicated by arrowheads) and cytoplasmic mRNA (magenta) of sygl-1 are visualized with spectrally distinct smFISH probes. DAPI marks DNA (blue). Maximum Z-projection is shown. Dashed line marks gonadal outline. Scale bar = 5 µm. Modified from Lee et al. (2016b).

Images taken using confocal microscopy do not require further image processing. However, images taken using wide-field (compound) microscopy must be deconvolved with carefully chosen image processing conditions (e.g., iterations). A caveat to the wide-field method is that over- or under-processed images can produce false-positive or false-negative signals. Using confocal images, the RNA spots can be systematically quantified with standard image processing (e.g., ImageJ) or published tools (e.g., MATLAB codes) (Mueller et al., 2013; Lee et al., 2016b). The wide-field microscope with a typical CCD camera (e.g., Hamamatsu C11440) generates microscopic images with the resolution high enough (pixel size: 0.1-0.2 µm) for identifying single RNA spots (Lee et al., 2016a). For confocal microscopy, we recommend using a pixel size similar to the size that works for wide-field microscopy (Abbaszadeh and Gavis, 2016; Lee et al., 2016b). As a first step of smFISH analysis, we quantify the size, shape (e.g., Gaussian distribution), signal intensities and subcellular location of detected RNA spots to validate singularity of detected RNA spots. In the Kimble lab, we consistently see nuclei containing one to four active transcription sites using intron-specific smFISH probes, with four matching the maximum number of loci of a gene (after DNA replication in S phase). The nascent RNAs are nuclear and colocalized to DAPI (or other DNA strain of choice). The mRNAs are cytoplasmic. Detected mRNA spots show tight signal distribution (C.V. < 0.5) in most RNA species, indicating that the detected spots are likely single RNA molecules (Lee et al., 2013 and 2016b). Control experiments such as detecting RNAs in the target gene knock-out background and using a different set of probes hybridizing the same target gene should be done to ensure the specificity of the probes to the target gene.


  1. Day 1, probe design notes
    We used the standard settings of smFISH probe designer (Biosearch Technologies), which spread the probes throughout the gene. We have not tested whether smFISH performance changes depending on the region the probes bind, but one prediction is that 5’ probes will better visualize earlier transcription events. RNA-seq data from consortiums (e.g., ModEncode) may provide information on the gene expression level before choosing the target gene. For the C. elegans germline, we use RNA-seq data (Ortiz et al., 2014 and Wormbase), in-situ hybridization data (NEXTDB) and qPCR to estimate the level of target gene expression.

  2. Day 1, dissection notes
    Periodically re-wipe gloves and pipettors with RNaseZap®. Use RNase-free filtered tips and RNase-free tubes; keep tip boxes closed when not in active use. Put 50-100 worms in the same tube for the entire protocol.

  3. Day 2, hybridization notes
    1. Minimize exposure to light during and after hybridization. Shield tube in your hand while changing buffers/tips, and wrap in foil for washes. To remove liquid from samples, use a P1000 pipettor because it can remove most liquid (within ~20 μl) without exposing tube to dissection scope light. The exceptions are before adding HB and before adding mounting media (look under the dissection scope [or check your tip before expelling liquid] and use a P1000, then a P200 and then a P10 to remove as much wash buffer as possible).
    2. Probe dilution is an effective way to optimize and should be determined empirically for each probe set. Probe stocks (250 μM) are diluted in RNase-free TE buffer before adding to the HB and typical final concentrations in HB are 0.025-0.5 μM (usually 0.25 μM is the first concentration we try). More dilute = less background.
    3. While samples are equilibrating in wash buffer, mix HB and probe in a separate RNase-free tube. It is recommended to use 50-100 μl HB + 1-3 µl probe per sample of fixed dissected worms.
      Note: Adding HB and probe sequentially on top of fixed germlines can also work, but mix gently and thoroughly–no vortexing.

  4. Day 3 notes
    1. Washing and mounting notes
      Note: With the Quasar 570 probe, either ProLong Gold Antifade or GLOX mounting media can be used. We saw essentially no difference in signal quality between the two media.
      1. Using ProLong Gold Antifade Mountant (PLG, Life Technologies):
        1. Slides and coverslips can be baked as an extra precaution against RNase contamination, or simply dedicate a box to smFISH.
        2. Collect any gonads/PLG remaining in tube and add to drop of gonads/PLG on the slide. Or if there is a lot left, mount on a separate slide.
        3. Remove bubbles from PLG droplet, being careful not to take gonads with them. Recommended methods include: popping with eyelash tool or flamed hot platinum wire, or aspirating gently with P10. Arrange worms inside drop quickly, using a pick to spread samples evenly.
        4. Cover with a 22 x 22 mm square coverslip. Place in drawer or other dark areas, on flat surface, at RT to cure. Put gentle pressure on coverslip to remove excess liquid and flatten gonads.
        5. Seal coverslip edges with nail polish. Stored in the dark at 4 °C, samples last for months.
      2. Using GLOX buffer for sensitive fluorophores (e.g., Quasar 570 and Quasar 670)
        1. Remove wash buffer, resuspend in 850 μl GLOX without enzymes. Let sit for 5 min (or place at 4 °C several hours until ready to image, as this method images each sample immediately after adding GLOX + enzymes).
        2. Immediately before imaging, remove buffer and add 100 μl GLOX + enzymes.
        3. Dissected gonads are extremely sticky; it is helpful to pipette up and down once in 1-10% Triton X-100 to coat the inside of the pipette.
        4. Pipette 4 μl sample onto a 22 x 22 mm square coverslip.
        5. Place a 12 x 12 mm square coverslip over sample drop. Lay a Kimwipe over the sample and press lightly on edges to remove excess liquid.
        6. Invert coverslip ‘sandwich’ so that the 12 x 12 coverslip is face down and placed on microscope slide to create imaging chamber. Seal with VALAP, starting with corners and then sealing edges. Be very careful that VALAP seal is airtight.
    2. Imaging notes
      Note: In case of using multiple fluorophores, carefully select the dichroic mirrors (wide-field microscope) or acquisition wavelength range (confocal microscope) to avoid signal bleed-through. Use settings to minimize photobleaching for image analysis and single-molecule quantitation. We recommend acquiring images sequentially, channel by channel, starting from the excitation light with the longest wavelength for the best performance. Below are the general smFISH image acquisition settings used in the Kimble lab.
      1. Microscope: Leica SP8 confocal
        Objective: 63x 1.4
        Zoom 300% to focus on distal ~60-70 µm of germline.
        Frequency: 400 Hz
        Laser power: between 1-5%, typically 3.5%
        Gain: typically 40 for HyD detectors
        8-16 line averaging, no accumulation
        Pinhole: ~1 Airy unit. Match pinholes for exon and intron channels to improve colocalization. We have adjusted to between 1.0 and 1.5 to gather more light–typically, we stay below 1.25-1.3.
        Bidirectional X: ON, phase = -32.40
        Z-stack interval = 0.3 μm
        Note: This way transcription sites are present in more than one slice and all mRNAs should be captured.
      2. Identifying C. elegans gonads: The gonads are typically attached to the worm carcass but sometimes they fall off during the wash step. Both attached and detached gonads can be imaged for smFISH. The extruded gonad is an elongate light-colored tissue with hundreds of packed small cells. The extruded intestine, by contrast, is an elongate dark-colored tissue with only tens of large cells and is therefore morphologically different from the gonad. It is not necessary to isolate the gonads but the microscopic field can be adjusted so that only the region of interest within the gonad will be imaged.


  1. Fixation: nuclease-free
    1. 1x PBS + 0.1% Tween-20 (PBSTw) (need 5 ml per sample)
      50 ml 1x PBS, nuclease-free
      50 μl Tween-20
    2. 1x PBSTw + 0.25 mM levamisole
      1 ml 1x PBS, nuclease-free
      1 µl Tween-20
      1 µl 0.25 M levamisole stock solution (51 mg in 1 ml M9 or 1x PBS)
    3. 1x PBSTw + 3.7% formaldehyde (need 1 ml per sample, make fresh)
      900 µl PBSTw
      100 μl 37% formaldehyde
    4. 1x PBS + 0.1% Triton X-100 (need 1 ml per sample)
      9.9 ml 1x PBS, nuclease-free
      100 μl 10% Triton X-100, diluted in nuclease-free water
    5. 70% ethanol (need 1 ml per sample)
      3 ml 100% EtOH, nuclease-free
      7 ml H2O, nuclease-free

  2. Probe and hybridization: nuclease-free
    1. TE buffer (DEPC-treated)
      10 mM Tris base
      1 mM EDTA
      pH 8.0
    2. Formamide
      Aliquot and store at 4 °C, warm to RT before opening
    3. smFISH wash buffer (need 6 ml per sample)
      1 ml 20x SSC
      1 ml formamide
      8 ml DEPC water
      10 μl Tween-20
    4. smFISH wash buffer + DAPI (need 1 ml per sample)
      1 ml wash buffer
      1 μl DAPI (1 mg/ml, stored in the dark at 4 °C)
    5. Hybridization buffer (HB)
      *Note: 10% formamide is generally used. However, formamide concentration can be adjusted to modify stringency. If target RNA has high GC content, try increasing formamide to 15-20%, or up to 50%. If adjusting formamide concentration, match concentration in wash buffer.
      1 g dextran sulfate
      7.3 ml DEPC water (or up to 10 ml volume)
      1 ml 20x SSC
      1 ml formamide
      1. Combine dextran sulfate and DEPC-H2O, rock ~30 min until fully dissolved. Add 20x SSC and formamide; invert to mix. Store in 500 μl aliquots at -20 °C.
      2. *Other labs (see for example Lee et al., 2013) also add (per 10 ml HB):
         10 mg E. coli tRNA
         100 μl Vanadyl ribonucleoside complex (200 mM)
         40 μl BSA (nuclease-free) (50 mg/ml)
        We started with these reagents but gradually omitted them. If experiencing problems with background, try adding them back in one at a time (in order listed).

  3. Reagents for GLOX buffer/mounting medium: nuclease-free
    1. GLOX buffer, no enzymes (need 1 ml per sample)
      850 µl H2O, nuclease-free
      100 µl 20x SSC, nuclease-free
      40 µl 10% glucose, nuclease-free
      10 µl 1 M Tris, pH 8.0, nuclease-free [TE also works]
      Note: Prepare fresh immediately before use.
    2. GLOX buffer + enzymes
      100 µl GLOX buffer
      1 µl glucose oxidase, 3.7 mg/ml
      1 µl catalase
      1 µl 200 mM Trolox
      Note: Keep on ice or at 4 °C.
    3. 10% glucose
      5 g glucose (weighed out in nuclease-free way)
      50 ml nuclease-free H2O
      Note: Pass through a 0.2 µm syringe filter and store at 4 °C.
    4. Glucose oxidase, 3.7 mg/ml
      37 mg glucose oxidase (weighed out in nuclease-free way)
      167 µl 3 M sodium acetate, pH 5.5, nuclease-free
      10 ml H2O, nuclease-free
      Note: Divide into 100 µl aliquots and store at -20 °C.
    5. Catalase
      Note: Store in the dark at 4 °C. Vortex or pipet up and down before use but do not spin.
    6. 200 mM Trolox
      5 mg Trolox (weighed out in nuclease-free way)
      1 ml 100% ethanol
      Note: Store as 100 µl aliquots at -20 °C.
    7. VALAP
      Vaseline + lanolin + paraffin (1:1:1 w/w/w)
      Note: To use, melt at ~70 °C and apply with paintbrush or metal spatula.


This protocol has been adapted from Lee et al. (2016b). ESK was supported by the American Cancer Society–George F. Hamel Jr. Fellowship (PF-14-147-01-DDC). HSS was supported by an Ellison Medical Foundation Fellowship of the Life Science Research Foundation. TRL is supported by the National Science Foundation Graduate Research Fellowship Program (Grant No. DGE-1256259). JK is an Investigator of the Howard Hughes Medical Institute.


  1. Abbaszadeh, E. K. and Gavis, E. R. (2016). Fixed and live visualization of RNAs in Drosophila oocytes and embryos. Methods 98: 34-41.
  2. Crittenden, S. L., Seidel, H. S. and Kimble, J. (2017). Analysis of the C. elegans germline stem cell pool. Methods Mol Biol 1463: 1-33.
  3. Ji, N. and van Oudenaarden, A. (2012). Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos. WormBook: 1-16.
  4. Lee, C., Roberts, S. E. and Gladfelter, A. S. (2016a). Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH. Methods 98: 124-133.
  5. Lee, C., Sorensen, E. B., Lynch, T. R. and Kimble, J. (2016b). C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool. Elife 5.
  6. Lee, C., Zhang, H., Baker, A. E., Occhipinti, P., Borsuk, M. E. and Gladfelter, A. S. (2013). Protein aggregation behavior regulates cyclin transcript localization and cell-cycle control. Dev Cell 25(6): 572-584.
  7. Mueller, F., Senecal, A., Tantale, K., Marie-Nelly, H., Ly, N., Collin, O., Basyuk, E., Bertrand, E., Darzacq, X. and Zimmer, C. (2013). FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4): 277-278.
  8. Ortiz, M. A., Noble, D., Sorokin, E. P. and Kimble, J. (2014). A new dataset of spermatogenic vs. oogenic transcriptomes in the nematode Caenorhabditis elegans. G3 (Bethesda) 4: 1765-1772.
  9. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. and Tyagi, S. (2008). Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10): 877-879.


单分子RNA荧光原位杂交(smFISH)是使用针对靶RNA特异性的多个荧光标记寡核苷酸探针来观察个体RNA分子的技术(Raj等人,2008; Lee等,2016a)。 我们调整了这种技术可视化线虫整个成虫或其种系中的RNA,其能够同时记录活跃转录位点的新生转录物和细胞质中的成熟mRNA(Lee等,2013和2016b)。 在这里,我们描述针对秀丽隐杆线虫挤压性腺优化的smFISH程序,试剂和显微镜设置的每个步骤。
【背景】smFISH能够在体内直接和精确地定量mRNA。 此外,通过复用smFISH探针,可以在同一细胞中同时测定多个RNA物种。 以前的出版物在线虫中使用了smFISH,但是这些研究使用了宽视野显微镜,其通常具有较低的空间分辨率并需要额外的图像处理(例如,图像去卷积)(Ji和van Oudenaarden,2012)。 在这里,我们描述了针对秀丽隐杆线虫组织优化的smFISH程序。 每个步骤都是详细的,包括使用共焦显微镜来获得精确的测量。 我们的协议最大限度地减少了样品与样品的变异性,并允许精确定量mRNA和新生转录物。

关键字:活跃转录位点, mRNA, smFISH, sygl-1, 秀丽隐杆线虫, 性腺


  1. 手套
  2. RNase-free微量离心管(1.5ml)(Thermo Fisher Scientific,Applied Biosystems TM,目录号:AM12450)
  3. 显微镜幻灯片(Fisher Scientific,目录号:12-544-1)
  4. 高精度显微镜盖玻璃(Marienfeld-Superior,目录号:0107052;可通过Azer Scientific订购)
  5. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  6. 铝箔
  7. Scalpel(羽毛一次性手术刀#10)(Medex Supply,目录号:GRF-2975#10)或针(25 G)(BD,目录号:305125)
  8. 不含RNase的过滤提示(Mettler-Toledo,Rainin,目录号:17007957,17002927和17014361分别为20,200和1,000μl提示)
  9. 0.2微米注射器过滤器(EMD Millipore,目录号:SCGP00525)
  10. ℃。线虫。一些菌株由CGC提供( http://www.cgc.cbs。 umn.edu ,由NIH研究基础设施项目办公室资助[P40 OD010440])
  11. 与荧光团缀合的smFISH探针(LGC BioSearch Technologies, https://www.biosearchtech。 com/),请参见下面的探针存储和稀释
  12. RNaseZap ®(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9780)
  13. ProLong Gold Antifade Reagent Mountant(Thermo Fisher Scientific,Invitrogen TM ,目录号:P36930)
  14. 指甲油
  15. RNase-free 1x PBS(Fisher Scientific,目录号:BP24384)
  16. 吐温-20(Fisher Scientific,目录号:BP337-100)
  17. 左旋咪唑(或四唑咪唑)(Sigma-Aldrich,目录号:L9756-10G);储存于-20°C
  18. 37%甲醛(AMRESCO,目录号:0493-500ML)
  19. Triton X-100(Fisher Scientific,目录号:BP151-100)
  20. 乙醇(Acros Organics,目录号:615090010)
  21. Tris碱(Fisher Scientific,目录号:BP152-5)
  22. 0.5M EDTA,pH 8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9260G)
  23. SSC(20x)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9763)
  24. 甲酰胺,去离子(EMD Millipore,目录号:4610-100ML);储存在4°C;在开启前温热至室温
  25. DEPC水,无菌(EMD Millipore,目录号:9610-1L)
  26. 4',6-二脒基-2-苯基吲哚,二盐酸盐(DAPI)(Thermo Fisher Scientific,Invitrogen,目录号:D1306)
  27. 硫酸葡聚糖(VWR,目录号:97061-196)
  28. 葡萄糖,无核酸酶(Fisher Scientific,目录号:D16-500)
  29. 不含核酸酶的Tris,pH 8.0,(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9855G)
  30. 葡萄糖氧化酶(MP Biomedicals,目录号:02195196);储存于4°C
  31. 过氧化氢酶(Fisher Scientific,目录号:S25239A)
  32. Trolox(Acros Organics,目录号:218940050)
  33. 乙酸钠(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9740)
  34. 特敏碱(Sigma-Aldrich,目录号:E10521-10G);储存于-20°C
  35. 凡士林
  36. 羊毛脂
  37. 石蜡
  38. 固定:无核酸酶(参见食谱)
    1. 1x PBS + 0.1%Tween-20(PBSTw)
    2. 1x PBSTw + 0.25mM左旋咪唑
    3. 1x PBSTw + 3.7%甲醛
    4. 1x PBS + 0.1%Triton X-100
    5. 70%乙醇
      1. 探针和杂交:无核酸酶(参见食谱)
        1. TE缓冲区
        2. 甲酰胺
        3. smFISH洗涤缓冲液
        4. smFISH洗涤缓冲液+ DAPI
        5. 杂交缓冲液(HB)
          1. GLOX缓冲液/安装介质试剂:无核酸酶(见配方)
            1. GLOX缓冲液,无酶
            2. GLOX缓冲液+酶
            3. 10%葡萄糖
            4. 葡萄糖氧化酶3.7 mg/ml
            5. 过氧化氢酶
            6. 200毫克Trolox
            7. VALAP
                1. 设备

                  1. 移液器(例如,Mettler-Toledo,Rainin,型号:P10,P20,P200,P1000)
                  2. 玻璃培养皿(例如直径:5厘米)或用于解剖蠕虫的凹陷滑块
                  3. 旋转器(例如,BD,Clay Adams Nutator,型号:421105)
                  4. 微量离心机在室温(Eppendorf,型号:5415 D)
                  5. 37℃培养箱
                  6. Vortexer(例如,,Baxter,目录号:S8223-1)
                  7. 共焦显微镜(Leica Microsystems,型号:Leica TCS SP8)
                    1. 程序

                      注意:图1显示了协议的流程图。除非另有说明,所有旋转速度均为400 x g(2,000 rpm),持续30秒。

                      图1. C的smFISH协议流程图。 elegans gonad

                      1. 探测准备使用
                        可通过Biosearch Technologies网站( https://www.biosearchtech)设计和购买smFISH探针。 COM/)。选择与用于实验的微观系统相容的荧光团。对于多路复用探针,请仔细选择探针结合的荧光团,以消除图像采集期间通道之间的渗透。要储存smFISH探针,在pH 8.0(终浓度为250μM)下重悬于20μlTE缓冲液(参见食谱),并储存于-20°C。该溶液可以在TE缓冲液中稀释至适当的稀释度(例如1:10-1:100)以制备工作溶液。选择最适用于smFISH的稀释度(例如,最高的信噪比),并将稀释的探针溶液保持在-20°C的黑暗中。当用于smFISH时,冷冻探针溶液应在室温下解冻。在我们手中,多次冻融不影响smFISH的表现。不要煮沸探头。

                      2. 第1天:性欲解剖为smFISH
                        1. 用RNaseZap ®擦拭台面,手套和移液器。
                        2. 从含有0.1%Tween-20(PBSTw)的麻醉(例如,0.25mM左旋咪唑[或四马来酸])的PBS中,将生长板上的蠕虫从生长板中挑取到玻璃培养皿(或抑郁症载玻片)中。或者,通过麻醉将200-300μlPBSTw加入生长板中,并将吸管蠕虫吸入培养皿或抑郁片。 PBSTw的较大体积(1-2ml)也可用于将蠕虫移动到培养皿中。在全虫感染的情况下,跳过步骤B2-B3并进行到步骤B4。
                        3. 直接在培养皿中尽可能快地挤压性腺(<10分钟,包括旋转)。用#10手术刀或针头切开咽后或尾部(图2)(Crittenden等人,2017)。
                        4. 将管子将性腺挤压成1.5ml微量离心管。旋转并清除上清液。
                        5. 加入1ml固定溶液(最终的3.7%甲醛v/v,在1×PBS + 0.1%吐温-20)中,并在室温(RT)摇瓶上孵育至少15分钟(最长45分钟)。
                        6. 旋转样品。
                        7. 在1ml PBSTw中洗1次。
                        8. 在室温下,在1ml 1x PBS + 0.1%Triton X-100中孵育10分钟以透化
                        9. 用1ml PBSTw洗2次。
                        10. 重悬于1ml 70%EtOH中并在4℃下孵育16-18小时(过夜)。样品可以储存在70%EtOH中长达一周。

                          图2.性腺挤出使用手术刀或针头挤压性腺,切割头部(咽后)或尾巴(黑色虚线)。蠕虫的内部压力会使性腺容易挤出。图片:Maria Gallegos。

                          1. 第2天:杂交
                            1. 使清洗缓冲液新鲜,让甲酰胺在开放前进入RT。
                            2. 开启前将杂交缓冲液(HB)置于RT中。
                            3. 解冻稀释探针,防止光照。
                            4. 旋转在第1天制备的挤出性腺,除去乙醇
                            5. 加入1ml洗涤缓冲液,在室温下平衡5分钟。旋转并移除尽可能多的缓冲区。
                            6. 将1μl稀释的探针添加到每个样品100μlHB(最终探针浓度为0.025-0.5μM),轻轻摇动管(见第2天杂交说明)。
                            7. 将样品重新悬浮在HB中,用探针混匀
                            8. 在37°C孵育过夜,旋转并保护光线。

                              1. 第3天:洗涤并安装样品(均在室温下)
                                1. 加入1ml smFISH洗涤缓冲液,倒入混合。旋转并移除缓冲区。
                                  注意:如果由于HB粘度,蠕虫不能很好地沉淀,则此旋转可以在400-500 x g(2,200-2,400 rpm)下进行1.5分钟。
                                2. 用1ml洗涤缓冲液+1μg/ml DAPI在旋转器上洗涤30分钟(防光)
                                3. 用1ml洗涤缓冲液洗涤2次。
                                4. 从固定样品中移除尽可能多的洗涤缓冲液。
                                5. 将性腺重悬于固定介质(例如,ProLong Gold Antifade Mountant或GLOX缓冲液)中。
                                6. 让坐30分钟到几个小时。
                                7. 将12-15μl的皮管吸入幻灯片,盖上22 x 22盖玻片。清除多余的液体。
                                8. 至少在室温或4°C下彻夜治疗,最多4天。 ProLong Gold用户手册推荐使用"固化"过程(〜24小时),以获得最佳性能。我们已经观察到,固化过程确实会改善荧光信号
                                9. 用指甲油密封长期储存。
                                    1. 数据分析


                                      Notch转录激活。 SYGL-1蛋白是种系干细胞(GSC)维持的关键调节剂。用光谱不同的smFISH探针显现个体初生转录物(明亮的白色核斑点,黄色和品红色通道的覆盖物,由箭头指示)和细胞质mRNA(品红色)。 DAPI标记DNA(蓝色)。显示最大Z投影。虚线标记性腺大纲。刻度棒=5μm。从Lee等人修改。 (2016b)。

                                      使用共焦显微镜拍摄的图像不需要进一步的图像处理。然而,使用宽场(复合)显微镜拍摄的图像必须用仔细选择的图像处理条件(例如,迭代)去卷积。广域方法的一个注意事项是,过或未处理的图像可能会产生假阳性或假阴性信号。使用共聚焦图像,可以使用标准图像处理(例如,ImageJ)或已发布的工具(例如,MATLAB代码)(Mueller et al。,2013; Lee等人,2016b)。具有典型CCD相机(例如,Hamamatsu C11440)的宽视野显微镜产生具有足够高的分辨率(像素大小:0.1-0.2μm)的用于识别单个RNA斑点的显微图像(Lee 等,。,2016a)。对于共聚焦显微镜,我们建议使用与大面积显微镜相似尺寸的像素尺寸(Abbaszadeh和Gavis,2016; Lee等人,2016b)。作为smFISH分析的第一步,我们量化检测到的RNA斑点的大小,形状(例如,高斯分布),信号强度和亚细胞位置,以验证检测到的RNA斑点的奇异性。在Kimble实验室,我们始终看到使用内含子特异性smFISH探针含有一至四个活性转录位点的核,四个匹配基因的最大位点数(在S期的DNA复制后)。新生的RNA是核的,并且共定位于DAPI(或其他选择的DNA菌株)。 mRNAs是细胞质的。检测到的mRNA斑点在大多数RNA物种中显示紧密的信号分布(C.V. <0.5),表明检测到的斑点可能是单个RNA分子(Lee等人,2013和2016b)。应进行对照实验,例如检测目标基因敲除背景中的RNA并使用与靶基因杂交的不同组的探针,以确保探针对靶基因的特异性。


                                      1. 第1天探针设计笔记
                                        我们使用smFISH探针设计器(Biosearch Technologies)的标准设置,它将探针扩展到整个基因。我们还没有测试smFISH性能是否根据探针结合的区域而发生变化,但是一个预测是5'探针将更好地可视化早期的转录事件。来自联盟(例如,ModEncode)的RNA-seq数据可以在选择靶基因之前提供关于基因表达水平的信息。对于 C。 elegans 种系,我们使用RNA-seq数据(Ortiz等人,2014和Wormbase),原位杂交数据(NEXTDB)和qPCR估计靶基因表达水平
                                      2. 第1天,解剖笔记
                                        用RNaseZap ®定期重新擦拭手套和移液器。使用不含RNase的过滤嘴和无RNase的管;不使用时请保持提示框关闭。将50-100个蠕虫放在同一个管中,用于整个协议。

                                      3. 第2天,杂交笔记
                                        1. 在杂交期间和之后尽可能减少暴露于光照。改变缓冲液/尖端时,将护盾管放在手中,然后将其包裹在洗涤液中。为了从样品中除去液体,请使用P1000移液器,因为它可以去除大部分液体(在〜20μl内),而不会将管暴露于解剖范围内。在添加HB之前和添加安装介质之前(请查看解剖范围[或在排出液体之前检查您的提示)并使用P1000,然后使用P200,然后P10删除尽可能多的洗涤缓冲液。 />
                                        2. 探针稀释是一种有效的优化方法,应根据每个探针组的经验确定。在添加到HB之前,将探针库(250μM)稀释在无RNase的TE缓冲液中,并且HB中的典型终浓度为0.025-0.5μM(通常为我们尝试的第一浓度为0.25μM)。更稀的=较少的背景
                                        3. 当样品在洗涤缓冲液中平衡时,将HB和探针混合在不含RNA酶的管中。建议每个固定解剖蠕虫样本使用50-100μlHB + 1-3μl探针。

                                          1. 第3天笔记
                                            1. 洗涤和安装笔记
                                              注意:使用Quasar 570探头,可以使用ProLong Gold Antifade或GLOX安装介质。我们看到两台媒体之间的信号质量基本没有差别
                                              1. 使用ProLong Gold Antifade Mountant(PLG,Life Technologies):
                                                1. 可以将幻灯片和盖玻片作为对RNase污染的额外预防措施进行烘烤,或者只是将盒子专门用于smFISH。
                                                2. 收集管中剩余的任何性腺/PLG,并在幻灯片上添加一系列性腺/PLG。或者如果还剩下很多东西,请放在单独的幻灯片上。
                                                3. 从PLG液滴中去除气泡,注意不要与其接触。推荐的方法包括:用睫毛工具或火焰热的铂金丝,或用P10轻轻吸气。安排蠕虫内滴快速,使用挑选均匀传播样品。
                                                4. 盖上一个22 x 22毫米方形的盖玻片。放在抽屉或其他黑暗的地方,在平坦的表面,在室温固化。对盖玻片施加温和的压力,以清除多余的液体并使性腺变平。
                                                5. 用指甲油密封盖玻片边缘。在黑暗中存储在4°C,样品持续数月。
                                                  1. 使用GLOX缓冲液作为敏感荧光团(例如,Quasar 570和Quasar 670)
                                                    1. 取出洗涤缓冲液,重新悬浮在不含酶的850μlGLOX中。放置5分钟(或放置在4℃几个小时,直到准备好成像,因为这种方法在加入GLOX +酶后立即对每个样品进行成像)。
                                                    2. 在成像之前,清除缓冲液并加入100μlGLOX +酶
                                                    3. 解剖性腺特别粘稠;在1-10%Triton X-100中上下移动一次以覆盖移液管内部是有帮助的。
                                                    4. 将4μl样品移至22 x 22 mm方形盖玻片上。
                                                    5. 在样品瓶上放置一个12 x 12 mm的盖玻片。将Kimwipe放在样品上,轻轻按压边缘以除去多余的液体。
                                                    6. 倒置盖玻片'三明治',使12×12盖玻片面朝下并放置在显微镜载玻片上以形成成像室。用VALAP密封,从角落开始,然后密封边缘。要非常小心,VALAP密封是气密的。
                                                        1. 影像笔记
                                                          1. 显微镜:徕卡SP8共焦
                                                            目标:63x 1.4
                                                            频率:400赫兹 激光功率:1-5%,通常为3.5%
                                                            增益:HyD探测器通常为40 8-16线平均,无积累
                                                            针孔:〜1个通风单位。匹配外显子和内含子通道的针孔,以改善共定位。我们调整到1.0和1.5之间以收集更多的光 - 通常,我们保持在1.25-1.3以下。
                                                            双向X:ON,phase = -32.40
                                                          2. 识别C.性腺性腺:性腺典型地附着在蠕虫尸体上,但有时它们在洗涤步骤中脱落。附属和分离的性腺都可以成像为smFISH。挤出的性腺是一个细长的浅色组织,有数百个包装的小细胞。相比之下,挤出的肠是细长的深色组织,只有几十个大细胞,因此在形态上与性腺不同。不需要分离性腺,但可以调节微观场,使得只有性腺中感兴趣的区域才能被成像。
                                                                1. 食谱

                                                                  1. 固定:无核酸酶
                                                                    1. 1x PBS + 0.1%Tween-20(PBSTw)(每个样品需要5ml)
                                                                      50毫升1x PBS,无核酸酶的
                                                                    2. 1x PBSTw + 0.25mM左旋咪唑
                                                                      1毫升1x PBS,无核酸酶
                                                                      1μl0.25M左旋咪唑储备溶液(51mg,在1ml M9或1x PBS中)
                                                                    3. 1×PBSTw + 3.7%甲醛(每样品需1ml,使新鲜)
                                                                    4. 1x PBS + 0.1%Triton X-100(每样品需1ml) 9.9 ml 1x PBS,无核酸酶的
                                                                      100μl10%Triton X-100,稀释在无核酸酶的水中
                                                                    5. 70%乙醇(每样品需1ml) 3 ml 100%EtOH,不含核酸酶的
                                                                      7毫升H 2 O,无核酸酶的

                                                                      1. 探针和杂交:无核酸酶
                                                                        1. TE缓冲(DEPC处理)
                                                                          10mM Tris碱基
                                                                          1 mM EDTA
                                                                          pH 8.0
                                                                        2. 甲酰胺
                                                                        3. smFISH洗涤缓冲液(每样品需6ml)
                                                                          1 ml 20x SSC
                                                                          8ml DEPC水
                                                                        4. smFISH洗涤缓冲液+ DAPI(每样品需1ml)
                                                                          1 ml洗涤缓冲液
                                                                          1μlDAPI(1 mg/ml,4℃黑暗保存)
                                                                        5. 杂交缓冲液(HB)
                                                                          7.3ml DEPC水(或最多10ml体积)
                                                                          1 ml 20x SSC
                                                                          1. 将硫酸葡聚糖和DEPC-H 2 O结合,摇匀30分钟,直到完全溶解。加入20x SSC和甲酰胺;反转混合在-20°C储存500μl等分试样。
                                                                          2. 其他实验室(参见例如Lee等人,2013)也添加了(每10 ml HB):
                                                                            40μlBSA(无核酸酶)(50 mg/ml)

                                                                              1. GLOX缓冲液/载体介质试剂:无核酸酶
                                                                                1. GLOX缓冲液,无酶(每样品需1ml)
                                                                                  850μlH 2 O,不含核酸酶的
                                                                                  100μl20x SSC,无核酸酶
                                                                                  10μl1 M Tris,pH 8.0,无核酸酶[TE]工作]
                                                                                2. GLOX缓冲液+酶
                                                                                  1微升葡萄糖氧化酶,3.7毫克/毫升 1μl过氧化氢酶
                                                                                  1μl200 mM Trolox
                                                                                3. 10%葡萄糖
                                                                                  50ml无核酸酶的H 2 O
                                                                                4. 葡萄糖氧化酶3.7 mg/ml 37 mg葡萄糖氧化酶(以无核酸酶的方式称重)
                                                                                  167μl3 M醋酸钠,pH 5.5,不含核酸酶的
                                                                                  10毫升H 2 O,不含核酸酶的
                                                                                5. 过氧化氢酶
                                                                                6. 200毫克Trolox
                                                                                  5 mg Trolox(以无核酸酶的方式称重)
                                                                                  1 ml 100%乙醇 注意:在-20°C下保存为100μl等分试样。
                                                                                7. VALAP
                                                                                  凡士林+羊毛脂+石蜡(1:1:1 w/w/w)
                                                                                    1. 致谢

                                                                                      该协议已经从Lee等人改编。 (2016B)。 ESK得到了美国癌症协会George F. Hamel Jr.奖学金(PF-14-147-01-DDC)的支持。 HSS得到埃里森医学基金会生命科学研究基金会奖学金的支持。 TRL由国家科学基金研究生研究奖学金计划(拨款号DGE-1256259)支持。 JK是霍华德休斯医学研究所的调查员。


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Copyright Lee et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lee, C., Seidel, H. S., Lynch, T. R., Sorensen, E. B., Crittenden, S. L. and Kimble, J. (2017). Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans. Bio-protocol 7(12): e2357. DOI: 10.21769/BioProtoc.2357.
  2. Lee, C., Sorensen, E. B., Lynch, T. R. and Kimble, J. (2016b). C. elegans GLP-1/Notch activates transcription in a probability gradient across the germline stem cell pool. Elife 5.