Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells
利用单分子荧光原位杂交进行贴壁动物细胞中RNA的检测   

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Nov 2017
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Abstract

Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects single RNA molecules in individual cells. This technique employs multiple single-stranded, fluorescent labeled, short DNA probes that hybridize to target RNAs in fixed cells, allowing for both the quantification and localization of cytoplasmic and nuclear RNAs at the single-cell level and single-molecule resolution. Analyzing smFISH data provides absolute quantitative data of the number of cytoplasmic (“mature”) mRNAs, the number of nascent RNA molecules at distinct transcription sites, and the spatial localization of these RNAs in the cytoplasm and/or nucleoplasm.

Keywords: mRNA (mRNA), Transcription (转录), Fluorescence in situ hybridization (荧光原位杂交), Single molecule resolution (单分子分辨率), Fluorescence microscopy (荧光显微技术), Adherent cells (贴壁细胞)

Background

Regulation of gene expression is one of the key determinants of cell fate and behavior. A major parameter of gene expression is mRNA level, which is determined by the rates of transcription and degradation. Therefore, measuring mRNA levels, as well as transcription and decay rates for particular transcripts (or all transcripts) has been the focus of numerous research projects.

Common molecular biology techniques, such as reverse transcription-PCR (RT-PCR), Northern blot analysis or RNA sequencing (RNA-Seq), typically require RNA extraction from the entire cell population. However, the results provide only a relative measure of mRNA content for the entire cell population, with a loss of single cell information. Single-cell RNA-Seq can provide more insight on the cell-to-cell variability of transcript levels. However, the current lower limit of detection is ~10 molecules/cell for a given RNA transcript (Svensson et al., 2017). RNA localization studies have shown that the spatial distribution of RNA in the cell can play a pivotal role in its function (Buxbaum et al., 2015), but the above-described methods lose that information in the process.

Single-molecule Fluorescence in situ Hybridization (smFISH) overcomes these limitations. In this method, the cells are first fixed and permeabilized. Then the cells are hybridized with a set of probes consisting of multiple short fluorescently labeled DNA oligonucleotides, which tile the length of the mRNA (Figure 1). The multiplicity of probes on a single RNA molecule increases the signal-to-noise ratio and allows for their detection by microscopy as diffraction-limited spots of similar intensity and dimensions. A 3D Gaussian fitting algorithm is used in image analysis tools to detect the spots in the images. smFISH can detect as little as a single RNA molecule and as much as several thousands. Importantly, smFISH provides spatial information of RNA localization in the cell. Although this protocol uses the example of mRNA, smFISH can be used to detect and quantify many types of RNA molecules, for example long non-coding RNAs (lncRNA) (Cabili et al., 2015), viral RNA genomes (Chou et al., 2013), ribosomal RNA (Buxbaum et al., 2014) and more.

There are two major disadvantages to smFISH. First, since the cells are fixed, smFISH cannot be used for temporal analysis of gene expression in the same cell (i.e., live imaging). Second, due to fluorophore limitations (i.e., only a small number of colors can be used for microscopy), smFISH is currently limited to study only 1-4 genes in a single experiment. However, multiple variations of smFISH exist leading to signal enhancement, increased resolution and/or multiplexing, and ultimately the simultaneous detection of transcripts from tens to hundreds of genes (reviewed at Buxbaum et al., 2015; Pichon et al., 2018). smFISH can be used in any organism, in cell culture and in tissue slices. Although the basic protocol concepts are similar, specialized protocols (which are abundant in the literature) are required for each sample type. Here we provide a detailed protocol for smFISH in adherent animal cells. smFISH originated in the lab of Prof. Robert H. Singer, which initially used a few (~5) 50-mer multiple-labeled probes (which were synthesized in-lab) for detection (Femino et al., 1998). Prof. Arjun Raj improved the method (Raj et al., 2008) by using a larger number of shorter single-label oligos (20-mer) that tile the entire length of the RNA. These protocols are available at their respective lab websites (e.g., Singer lab and Raj lab). However, these protocols are outdated (e.g., in regards to reagents and types of probes), and are lacking in details. There are published method papers for smFISH, but surprisingly only a few on adherent cells (e.g., Lee et al., 2016). Furthermore, many labs that use smFISH routinely develop in-house software for smFISH analysis. This is inefficient, confusing, and not very user-friendly to biologists that lack programming background.

This protocol was originally developed at the Singer lab (e.g., Haimovich et al., 2017) and it is presented here with minor modifications made at the Gerst lab. It is partially based on the Raj protocol and the Stellaris® RNA FISH protocol (see Biosearch technologies website). A major difference from other protocols is that we recommend use of the FISH-quant program (Mueller et al., 2013; Tsanov et al., 2016), which is user-friendly, and hope it will be used to standardize smFISH analysis.


Figure 1. A scheme depicting the main principle of smFISH: multiple fluorescently labeled probes tile the length of the mRNA

Materials and Reagents

  1. Pipette tips
  2. Microscope Glass slides 25 x 75 mm x 1 mm thick (e.g., Thermo Scientific, catalog number: 421-004T or equivalent)
  3. Glass coverslips, round, 18 mm, #1 (e.g., Thermo Scientific, catalog numbers: 11709875 or equivalent)
  4. 1.7 ml plastic tubes
  5. 15 ml plastic tubes
  6. Nuclease-free Barrier tips (10 µl, 200 µl, 1,000 µl)
  7. Hybridization chamber (e.g., closed plastic box, 15 cm tissue culture dish, Petri dish)
  8. Parafilm (Bemis, catalog number: PM996)
  9. Kimwipes (e.g., KCWW, Kimberly-Clark, catalog number: 34120 or equivalent)
  10. 12-well plates (e.g., Costar, catalog number: 3513 or equivalent)
  11. Aluminum foil
  12. Adherent cells of interest (e.g., mouse embryonic fibroblasts [MEFs], Gastric carcinoma NCI-N87 cells)
  13. Suitable culture media and supplements (e.g., DMEM supplemented with 10% FBS and penicillin/streptavidin)
  14. (Optional) Extracellular matrix substrate, e.g., Fibronectin (Sigma-Aldrich, catalog number: F1141-5mg)
  15. 70% ethanol
  16. Sterile PBS x1 pH 7.4, no calcium, no magnesium (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 10010-015 or equivalent)
  17. 10x PBS, no calcium, no magnesium (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 14200-067 or equivalent)
  18. MgCl2 (e.g., Sigma-Aldrich, catalog number: M8266-100G or equivalent)
  19. Glycine (e.g., Sigma-Aldrich, catalog number: G8898-500G or equivalent)
  20. 32% paraformaldehyde (PFA) (Electron Microscopy Sciences)
  21. Surfact-AmpsTM X-100 (Triton X-100) 10% solution (Thermo Scientific, catalog number: 28314)
    Note: This high-purity Triton X-100 gives the best results, but other Triton X-100 products will provide satisfactory results.
  22. 20x Saline-sodium citrate (SSC) buffer (e.g., Sigma-Aldrich, catalog number: S6639-1L or equivalent)
  23. Formamide (Sigma-Aldrich, catalog number: 47671-250ml or equivalent) (keep at 4 °C)
  24. Dextran sulfate (Sigma-Aldrich, catalog number: D6001 or equivalent)
  25. E. coli tRNA (100 mg) (Roche, catalog number: 10109541001) (keep at -20 °C)
  26. Bovine serum albumin (BSA) (20 mg/ml) (Roche, catalog number: 10711454001) (keep at -20 °C)
  27. Vanadyl ribonucleoside complex (VRC) 200 mM (e.g., Sigma-Aldrich, catalog number: 94742-1 ml or equivalent) (keep at -20 °C)
  28. Nuclease-free water
  29. DAPI (nuclear stain) (e.g., Sigma-Aldrich, catalog number: D9542-1mg or equivalent)
  30. Fluorescent oligo probe set (e.g., Stellaris probes against human HER2-Quasar570 (Biosearch technologies, DesignReady catalog number: VSMF-2102-5) (see Procedure A for design and production of probes) (keep at -20 °C)
  31. Anti-fade reagent (e.g., ProLong anti-fade series from Thermo scientific)
  32. (Optional) High-quality nail polish (e.g., Electron Microscopy Sciences, catalog number: 72180)
  33. Immersion oil 1.518, suitable for the microscope/objective
  34. PBSM buffer (see Recipes)
  35. Fixation buffer (see Recipes)
  36. Quenching buffer (see Recipes)
  37. Permeabilization buffer (see Recipes)
  38. Pre-hybridization (Pre-hyb) buffer (see Recipes)
  39. Hybridization buffer (see Recipes) (keep at -20 °C)
  40. Hybridization chamber (see Recipes)
  41. DAPI stain solution (see Recipes) (keep at 4 °C)

Equipment

  1. Pipet aid (recommended: S1 pipet filler, Thermo Fisher Scientific, catalog number: 9501)
  2. Tweezer, straight, pointed, stainless steel tip (e.g., Ideal-Tek, catalog number: 4 SA or equivalent)
  3. (Optional) Vacuum trap 
  4. Chemical (fume) hood
  5. Biological hood/biosafety cabinet (for cell culture work)
  6. Cell culture incubator suitable for cell culture of your choice (e.g., 37 °C, 5% CO2)
  7. 37 °C incubator (e.g., an incubator that is used to culture bacterial plates)
  8. Cardboard tray for slides (e.g., Thermo Fisher Scientific, catalog number: 12-587-10)
  9. Wide-field fluorescent microscope (e.g., Olympus, model: BX-61; Nikon, model: Eclipse Ti-E inverted fluorescence microscope or Zeiss, model: AxioObserver Z1) equipped with the following:
    1. Fluorescent light source [e.g., Illuminator HXP 120 V light source (Carl Zeiss, model: Illuminator HXP 120 V) or X-cite 120 PC lamp (Excelitas Technologies, X-Cite® 120PC)]
    2. Filter sets suitable for the fluorophores used + DAPI (blue) filter
    3. Automated motorized stage for sub-micron movement in X, Y, and Z axes [e.g., MS 2000 XYZ automated stage (ASI, model: MS 2000) or motorized XYZ scanning stage, 130x100 PIEZO (Zeiss, catalog number: 432027-9001-000)]
    4. Plan-Apo 100x (preferred) or 63x oil immersion objective with high NA (1.35 NA or more)
    5. CCD or sCMOS high-resolution digital camera [e.g., Flash 4 sCMOS (Hamamatsu) or Pixis 1024 CCD camera (Photometrics)]
    6. Software suitable to control the microscope (according to manufacturer) for automated imaging of multiple channels, multiple z-stacks and multiple fields (e.g., MetaMorph, ZEN2, µmanager)
  10. Computer capable of image processing (strong CPU, at least 32 GB RAM)
  11. Computer for data storage
    Data storage on computer or external drive to allow for storage of 10’s of GBs and up to TB’s of cumulative image data.

Software

  1. MATLAB–R2015a version or higher 
  2. FISH-quant (Mueller et al., 2013; Tsanov et al., 2016) (free software https://bitbucket.org/muellerflorian/fish_quant)
  3. ImageJ/FIJI (Schindelin et al., 2012) (free software https://imagej.net/Fiji)
  4. Stellaris FISH probe designer (https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer); requires a user account (free) 
  5. Excel or equivalent program

Procedure

  1. Design and labeling of oligonucleotide probes
    1. Probes are 18-22 mer DNA oligonucleotides that are fluorescently labeled with a fluorescent organic dye at one or both ends. The most common dyes used are the cyanine (Cy), Alexa, and Atto dye series. To design the probes, first obtain the RNA sequence of interest.
      Note: For good signal-to-noise (SNR) ratio that will allow detection of the FISH spots over the background, it is recommended to use at least 25 probes (best = ~48 probes) per transcript, which means that a short transcript (i.e., < ~500 nt) may not be suitable for this version of smFISH. Alternative methods such as smiFISH (Tsanov et al., 2016), RNAscope (Wang et al., 2012) or clampFISH (Rouhanifard et al., 2018; preprint), which enhance the FISH signal, might be more suitable for short RNAs. 
    2. We recommend using the Stellaris probe design web tool. Insert the sense strand sequence and choose the required parameters (i.e., organism, specificity level, number of probes, probe length, and minimal spacing). We recommend choosing the highest level of specificity, i.e., 5, and 48 probes of 20 nt with 2 nt spacing as default. If there are < 25 probes, parameters can be changed until you are satisfied.
    3. There are multiple protocols to label the probes. The simplest solution, which we recommend for consistency and ease, is to order the Stellaris RNA FISH probes from Biosearch technologies (https://www.biosearchtech.com/). However, these are relatively expensive. One alternative is to order DNA oligos with amine ends and label with an amine reactive dye (Singer, 1998). Note that this protocol is for 50 nt oligos, but can be utilized for 20 nt oligos. This method works well, but is still relatively expensive. A cheaper option is to enzymatically label the probes (Gáspár et al., 2017 and 2018). 
    4. The oligo probe set should be suspended in nuclease-free water (for Stellaris probes, prepare a 25 µM solution) and kept at -20 °C in the dark. Probes can be divided into aliquots of 10-20 µl to avoid multiple freeze-thaw cycles (although in our experience we did not detect any noticeable deterioration after multiple freeze-thaw cycles).

  2. Cell culture
    General comments: 
    1. Work in a bio-safety cabinet (biological hood) for sterile cell culture work.
    2. This protocol is designed for adherent cells. For non-adherent cells, it is required to add additional steps to adhere the cells to the coverslip. Users who plan such steps need to consider these points: 
      1. How to adhere the cells to the glass (e.g., by poly-lysine coating)? 
      2. Decide whether to adhere the cells to the glass before fixation or after the final wash step. Each case could necessitate different protocol steps (e.g., either to perform the washes on coverslips or in tubes, or perform fixation either before or after adherence) and might even yield different results. 
      3. Calibrate the number of cells per coverslip. 
    3. If possible, it is recommended to add an additional cell line as a negative control for the FISH probes used (e.g., knockout cells, cells of a different species that express the RNA of interest, but with a nucleotide sequence that has low homology, etc.). This is helpful both for calibrating the FISH signals for the specific probes, as well as for FISH spot analysis. It is preferable to verify that the knockout cell line does not express the RNA of interest. We note that truncated RNAs might be expressed from knockout cells and these can be detected by FISH.

    1. Pre-warm 1x PBS and culture media to 37 °C.
    2. (Optional) If coating the coverslips, prepare a coating solution (e.g., dilute fibronectin 1:100 in 1x PBS).
    3. For each sample, place a coverslip in a well of 12-well plate.
    4. Wash briefly with 1 ml of 70% ethanol. Aspirate ethanol.
    5. Wash briefly with 1 ml sterile 1x PBS.
    6. (Optional) Add 1 ml of coating solution and incubate as required (e.g., for fibronectin, incubate for 10-20 min in a cell culture incubator). Remove coating solution and wash with 1 ml PBS (x1).
    7. Place 1 ml culture media per well.
    8. Seed adherent cells of choice. 
    9. Culture the cells in a cell culture incubator for required time of the experiment; do not let the culture become confluent. Aim for a maximum of ~80%-90% confluence at the time of fixation.

  3. FISH
    General comments:
    1. To avoid RNase contamination of samples, wear gloves, use barrier tips and avoid working on surfaces where there is regular use of RNases (e.g., from plasmid prep kits). 
    2. For safety, work with PFA and formamide solutions should be performed in a chemical (fume) hood.
    3. All steps except “hybridization” are performed while the coverslips remain in the well, with 1 ml of solution added per well.
    4. It is recommended to pipet liquids on the wall of the well and not directly onto the cells. For cells with delicate structures (e.g., dendrites, membrane nanotubes), it is recommended to use a pipet aid at the slowest setting and not to use vacuum aspirator.
    5. There is no need to shake the 12-well plate during wash steps.

    1. Wash cells with PBSM (3 quick rinses).
    2. Fix cells by incubating with fixation buffer (prepared fresh) for 10 min (not longer, see Note 1) at room temperature (RT).
    3. Wash with quenching buffer, 10 min at RT. 
    4. Wash with PBSM for 10 min at RT. Repeat this step. Cells can be left overnight at 4 °C at this point.
    5. Permeabilize cells by incubating with permeabilization buffer for 10 min (not longer, see Note 1) at RT.
    6. Wash with PBSM for 10 min at RT. Repeat this step.
    7. Incubate with Pre-hyb buffer for 30 min at RT.
    8. While waiting (Step C7), mix the pre-made hybridization buffer with the probes, and prepare the hybridization chamber (see Recipes and Note 2).
    9. Place 45 µl of hybridization buffer at each intended coverslip position in the hybridization chamber. There is no need to remove large bubbles, but avoid small foam-like bubbles.
    10. With the tweezers, gently lift each coverslip from the well, remove excess liquid by touching the edge on a Kimwipe and place the coverslips with the cells facing down on the hybridization buffer (see Video 1 and Note 3).

      Video 1. Transferring coverslips from 12-well plate to hybridization chamber

    11. Seal the hybridization chamber with Parafilm, wrap with aluminum foil, and place in a 37 °C incubator for 3 h to overnight.
    12. Prepare a new (or same) 12-well plate with Pre-hyb buffer.
    13. Use the tweezers to transfer the coverslips back to the 12-well plate, cells facing up.
    14. Cover the plate with aluminum foil and incubate in the 37 °C incubator for 15 min.
    15. Wash again in Pre-hyb buffer, 15 min at 37 °C. 
    16. Quick rinse with 2x SSC at RT (3 quick rinses).
    17. Stain in DAPI stain solution (pre-warmed to RT) for 1 min at RT.
    18. Wash for 5 min with 2x SSC.
    19. During this final wash step, prepare microscope slides in the cardboard tray: 
      1. Label the slide(s).
      2. Just before lifting the coverslips, add 20 µl of Pro-Long anti-fade solution for each cover slip (there can be two per slide). Remove any air bubbles. See also Note 4.
    20. Use tweezers to lift coverslips, remove excess liquid and place cell-side facing down, on the Pro-Long anti-fade drop. 
    21. Let dry at RT in the dark for at least several hours (for best images, wait > 24 h). 
    22. (Optional) For long-term storage, seal with nail polish around the edges of the coverslip after the Pro-Long dries. 
    23. Slides can be stored at RT for several days (at least, we have not checked longer than a week). Keep at -20 °C for long-term storage (months to years). 

  4. Imaging
    Important: All slides from the same experiment should be imaged using the same exact conditions.
    1. Imaging can be performed on any wide-field microscope, as detailed in the Equipment section.
    2. Do not use a confocal microscope for smFISH imaging. The higher light intensity of the lasers can cause rapid bleaching of the FISH signal. Since the smFISH signal is relatively weak and requires long exposure times to collect enough light, photobleaching limits the total amount of light collected. This is particularly problematic when taking multiple z sections in order to create a 3D image stack. 
    3. Images should be taken at the relevant channels with descending color order [e.g., Cy5 (670 nm, far-red), Cy3 (570 nm, yellow-orange), Alexa488 (520, green), and DAPI (460 nm, blue)]. This is particularly important when imaging with DAPI, since we have noticed that in some cell types (e.g., MEFs, HEK293T cells), imaging through the DAPI channel may cause the appearance of granular autofluorescence in other channels. This was infrequent (i.e., it did not happen every time) but it was reproduced using several microscopes.
    4. Exposure time: For FISH, it is recommended to use maximum (100%) power of the light source and an exposure time of 1-3 s for each FISH channel. For DAPI, use a low power setting and very short exposure time (e.g., 30% power, 20-40 ms).
    5. For FISH spot detection, more photons (i.e., longer exposure time) means better detection, but also increased background fluorescence. Therefore, the user should adjust the time and exposure parameters accordingly.
    6. Z-sections: In order to detect RNA spots throughout the volume of the cell, multiple z-sections should be collected. It is recommended to use 0.2-0.3 µm steps and collect at least 30 sections (i.e., for flat cells, like fibroblasts) or more (e.g., for HEK293T or N87 cells we use 41 z-sections). When imaging, choose the option to image all z-sections in one channel before switching to the next channel.
    7. Binning: For better resolution, use pixel binning 1 x 1. However, sometimes for the benefit of signal enhancement over resolution, a 2 x 2 binning may be used.
    8. For examples of FISH images and z-stacks, see Figure 2 and Videos 2-5.


      Figure 2. Examples of unfiltered and filtered FISH images produced by FISH-quant. smFISH on human NCI-N87 gastric carcinoma cells (top row) and immortalized MEFs (bottom row) were performed using Stellaris probes against human HER2 mRNA. Imaging was performed on a Zeiss AxioObserver Z1 DuoLink dual camera imaging system equipped with Illuminator HXP 120 V light source, PlanApo 100x 1.4 NA oil immersion objective and Hamamatsu Flash 4 sCMOS cameras. For both cell types, 41 steps of 0.2 μm z-stack images were taken using a motorized XYZ scanning stage 130 x 100 PIEZO, and ZEN2 software at 0.0645 μm/pixel. Images show a maximum projection of middle z-sections. Note that the brightness of the MEFs FISH image (lower left) was increased compared to that of N87, to allow better visualization of the cells. See Videos 2-5 for the full z-stack of each image. Scale bars = 10 µm.

      Video 2. 3D FISH image of N87 cell with human HER-Q570 probes

      Video 3. 3D filtered image of the cell in Video 2

      Video 4. 3D FISH image of MEF cell with human HER-Q570 probes

      Video 5. 3D filtered image of the cell in Video 4

Data analysis

FISH spot analysis is performed by programs that fit the diffraction-limited spots using a 3D Gaussian fitting algorithm. Many labs produce their own software/scripts. We recommend FISH-quant (Mueller et al., 2013; Tsanov et al., 2016) since it is user-friendly and can perform multiple analyses (i.e., mature mRNA, transcription site analysis, co-localization). The images for FISH-quant need to be in TIFF format as a separate file of multiple z-sections obtained for each channel. If the microscope does not save files in TIFF format, convert the images by using another program (e.g., FIJI). Follow the instructions for FISH-quant to perform analysis.
Briefly:

  1. Install MATLAB and then install FISH-quant.
  2. In the main interface of FISH-quant:
    1. Choose the folders (main folder, images folder, outlines folder, results folder).
    2. Insert the experimental parameters: XY pixel size (based on camera and binning), Z pixel size, refractive index of the oil, NA of the objective, excitation and emission wavelengths of the FISH probe fluorophore. 
    3. Under Tools → outline designer: draw outlines for cells, nuclei and transcription sites (TS) (hint: use the automatic “detect nucleus” and “TS auto detect” based on the DAPI and TS/FISH staining, respectively). 
    4. Upload outline of image.
    5. Filter the background (try different modes and parameters for best filtering). For examples of filtered images and z-stacks, see Figure 2 and Videos 3 and 5.
    6. Perform pre-detection according to instructions.
    7. Fit spots using the Gaussian algorithm.
    8. Use thresholding parameters to achieve the best results.
    9. Save the detection settings.
  3. From Tools → Batch processing it is possible to perform analysis of multiple images. The thresholding parameters can be modified after batch analysis to fine-tune the results. It is recommended to set the parameters so that a negative control will give close to 0 spots. 
  4. Use Tools → Spot inspector to eliminate obvious false positives (e.g., areas of high autofluorescence). 
  5. The data from FISH-quant is saved as a ‘.txt’ file. The data can be copied and pasted to Excel for further analysis (e.g., Figure 3).


    Figure 3. FISH-quant analysis of human HER2 mRNA expression level in human N87 cancer cells and MEFs. Each circle/triangle represents the number of spots scored for a single cell.

Notes

  1. The most critical incubation times to keep are at the fixation and permeabilization steps. For all other wash steps, samples can be left for longer incubation times. From our experience, 30 min for quenching/wash and up to 60 min for Pre-hyb did not have any adverse effects on the FISH. Shorter incubation times (e.g., 5 min) or fewer washes (e.g., once instead of twice after fixation/permeabilization) had a small, but distinct, adverse effect. Shortening post-hybridization wash times will lead to a significant increase in the background signal. The incubation times in this protocol have worked well for many cell types (including MEFs, many cancer cell lines, mouse primary hippocampal neurons, and others). However, incubation times may require optimization by users to suit their own cell lines.
  2. There is no need to dim the lights when working with the fluorophore, but it is recommended to minimize light exposure until the addition of the anti-fade solution. The room should be darkened during imaging to minimize autofluorescence and to avoid background light contamination. 
  3. Use gentle force to grab and lift the coverslips with the tweezers, since coverslips can break easily. If the coverslip breaks to two large pieces, it is still salvageable and the experiment can continue (make sure to separate the two pieces so they do not stick one on top of the other). Furthermore, if the tweezers do not hold the coverslips firmly, the coverslips might fall to the bench. Again, the coverslip is still salvageable, but the user needs to guess the correct side of the cells.
  4. There are other commercially available anti-fade solutions, as well as lab-made anti-fade solutions that can be used (e.g., by using glucose oxidase). However, we have no experience with those, and these might not be suitable for FISH or long-term storage.

Recipes

  1. PBSM buffer (500 ml)
    50 ml 10x PBS
    2.5 ml MgCl2 1 M
    475 ml nuclease-free water
    Store at RT
  2. Fixation buffer (8 ml)
    7 ml PBSM
    1 ml PFA 32%
    Prepare fresh
  3. Quenching buffer (10 ml)
    9.5 ml PBSM
    0.5 ml 2 M glycine
    Prepare fresh as it tends to get microorganism contaminations
    Prepare stock solutions in advance: 
    1. PBSM buffer (Recipe 1)
    2. 2 M glycine stock solution in water, filter sterilize (store at RT)
  4. Permeabilization buffer (100 ml)
    10 ml 10x PBS
    1 ml Surfact-AmpsTM X-100 10%
    89 ml nuclease-free water
    Store at RT
  5. Pre-hybridization (Pre-hyb) buffer (10 ml)
    1 ml 20x SSC
    1 ml Formamide (warm to room temperature before use)
    8 ml nuclease-free water
    Prepare fresh
  6. Hybridization buffer 

    Add probes fresh to hybridization buffer. It is recommended to calibrate probe concentration when using probes for the first time (default: 250 nM or 10 ng/sample).
    Prepare stock solutions in advance:
    1) 10 mg/ml E. coli tRNA solution in nuclease-free water. Store at -20 °C.
    2) 20% Dextran sulfate in water (viscous, takes 30-60 min to dissolve completely). Store at RT.
  7. Hybridization chamber
    1. Plastic box or plastic dish (e.g., Petri dish, 15 cm tissue culture dish). The size of the chamber should accommodate all the coverslips allowing for at least a few millimeters between the coverslips
    2. Place parafilm on the bottom of the chamber. Avoid wrinkles where the coverslips will be placed
    3. (Optional) Draw a grid (each square should fit a single coverslip) and label the squares
    4. Take the cap of a 15 ml conical tube and place it at the edge of the chamber. Fill the cap with 1 ml of water or buffer (this is required to maintain humidity in the chamber)
    5. Prepare a piece of parafilm to seal the chamber after coverslips are placed
    6. Prepare aluminum foil to cover the chamber to protect from light
    7. The plastic chamber can be re-used multiple times
  8. DAPI stain solution (200 ml)
    20 ml 20x SSC
    10 µl of 10 mg/ml DAPI (final concentration: 0.5 µg/ml)
    180 ml nuclease-free water
    Store at 4 °C in the dark
    Prepare DAPI stock solution in advance:
    10 mg/ml DAPI solution in water. Store at -20 °C in the dark

Acknowledgments

G.H. is a recipient of the Koshland Foundation and McDonald-Leapman Grant Senior Post-doctoral fellowship. This work was funded by grants from the Joel and Mady Dukler Fund for Cancer Research (WIS), a Proof-of-Principle Grant from the Moross Integrated Cancer Center, Weizmann Institute, and US-Israel Binational Science Foundation-National Science Foundation (#2015846) to J.E.G.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

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  3. Cabili, M. N., Dunagin, M. C., McClanahan, P. D., Biaesch, A., Padovan-Merhar, O., Regev, A., Rinn, J. L. and Raj, A. (2015). Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol 16: 20.
  4. Chou, Y. Y., Heaton, N. S., Gao, Q., Palese, P., Singer, R. H. and Lionnet, T. (2013). Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis. PLoS Pathog 9(5): e1003358.
  5. Femino, A. M., Fay, F. S., Fogarty, K. and Singer, R. H. (1998). Visualization of single RNA transcripts in situ. Science 280(5363): 585-590.
  6. Gáspár, I., Wippich, F. and Ephrussi, A. (2017). Enzymatic production of single-molecule FISH and RNA capture probes. RNA 23(10): 1582-1591.
  7. Gáspár, I., Wippich, F. and Ephrussi, A. (2018). Terminal deoxynucleotidyl transferase mediated production of labeled probes for single-molecule FISH or RNA capture. Bio-protocol 8(5): e2750.
  8. Haimovich, G., Ecker, C. M., Dunagin, M. C., Eggan, E., Raj, A., Gerst, J. E. and Singer, R. H. (2017). Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells. Proc Natl Acad Sci U S A 114(46): E9873-E9882.
  9. Lee, C., Roberts, S. E. and Gladfelter, A. S. (2016). Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH. Methods 98: 124-133.
  10. 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.
  11. Pichon, X., Lagha, M., Mueller, F., and Bertrand, E. (2018). A growing toolbox to image gene expression in single cells: sensitive approaches for demanding challenges. Mol Cell 71(3): 468-480.
  12. 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.
  13. Rouhanifard, S. H., Mellis, I. A., Dunagin, M., Bayatpour, S., Symmons, O., Cote, A. and Raj, A. (2018). Exponential fluorescent amplification of individual RNAs using clampFISH probes. bioRxiv 222794.
  14. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  15. Singer, R. H. (1998). Preparation of probes for in situ hybridization. https://www.einstein.yu.edu/uploadedFiles/LABS/robert-singer-lab/probe_prep.pdf.
  16. Svensson, V., Natarajan, K. N., Ly, L. H., Miragaia, R. J., Labalette, C., Macaulay, I. C., Cvejic, A. and Teichmann, S. A. (2017). Power analysis of single-cell RNA-sequencing experiments. Nat Methods 14(4): 381-387.
  17. Tsanov, N., Samacoits, A., Chouaib, R., Traboulsi, A. M., Gostan, T., Weber, C., Zimmer, C., Zibara, K., Walter, T., Peter, M., Bertrand, E. and Mueller, F. (2016). smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res 44(22): e165.
  18. Wang, F., Flanagan, J., Su, N., Wang, L. C., Bui, S., Nielson, A., Wu, X., Vo, H. T., Ma, X. J. and Luo, Y. (2012). RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14(1): 22-29.

简介

转录和RNA衰变在基因表达过程中起关键作用,并且准确测量细胞mRNA水平的能力对于理解该调节是必不可少的。 在这里,我们描述了单分子荧光原位杂交(smFISH)方法(如Haimovich et al。,2017中所进行的),其检测单个细胞中的单个RNA分子。 该技术使用多个单链,荧光标记的短DNA探针,其与固定细胞中的靶RNA杂交,允许在单细胞水平和单分子分辨率下定量和定位细胞质和核RNA。 分析smFISH数据提供了细胞质(“成熟”)mRNA数量,不同转录位点的新生RNA分子数量以及这些RNA在细胞质和/或核质中的空间定位的绝对定量数据。

【背景】 基因表达的调节是细胞命运和行为的关键决定因素之一。基因表达的主要参数是mRNA水平,其由转录和降解速率决定。因此,测量mRNA水平,以及特定转录物(或所有转录物)的转录和衰变速率一直是许多研究项目的焦点。

常见的分子生物学技术,例如逆转录-PCR(RT-PCR),Northern印迹分析或RNA测序(RNA-Seq),通常需要从整个细胞群体中提取RNA。然而,结果仅提供了整个细胞群的mRNA含量的相对量度,同时丧失了单细胞信息。单细胞RNA-Seq可以提供更多关于转录水平的细胞间变异性的见解。然而,对于给定的RNA转录物,目前检测的下限是~10个分子/细胞(Svensson 等人,,2017)。 RNA定位研究表明,细胞内RNA的空间分布在其功能中起着关键作用(Buxbaum et al。,2015),但上述方法在此过程中失去了信息。 。

单分子荧光原位杂交(smFISH)克服了这些限制。在该方法中,首先将细胞固定并透化。然后将细胞与一组探针杂交,所述探针由多个短荧光标记的DNA寡核苷酸组成,其平铺mRNA的长度(图1)。单个RNA分子上的多个探针增加了信噪比,并允许通过显微镜检测它们作为具有相似强度和尺寸的衍射限制斑点。在图像分析工具中使用3D高斯拟合算法来检测图像中的斑点。 smFISH可以检测到单个RNA分子和数千个RNA分子。重要的是,smFISH提供细胞中RNA定位的空间信息。尽管该方案使用mRNA的例子,但smFISH可用于检测和定量许多类型的RNA分子,例如长的非编码RNA(lncRNA)(Cabili et al。,2015),病毒RNA基因组(Chou et al。,2013),核糖体RNA(Buxbaum et al。,2014)等。

smFISH有两个主要缺点。首先,由于细胞是固定的,因此smFISH不能用于同一细胞中基因表达的时间分析(即,实时成像)。其次,由于荧光团的限制(即,只有少量颜色可用于显微镜检查),smFISH目前仅限于在单个实验中仅研究1-4个基因。然而,存在多种smFISH变异导致信号增强,分辨率增加和/或多重化,并最终同时检测数十至数百个基因的转录本(综述于Buxbaum et al。,2015; Pichon et al。,2018)。 smFISH可用于任何生物体,细胞培养物和组织切片中。虽然基本协议概念是相似的,但每种样本类型都需要专门的协议(文献中很丰富)。在这里,我们提供了粘附动物细胞中smFISH的详细方案。 smFISH起源于Robert H. Singer教授的实验室,该实验室最初使用了一些(~5)50-mer多标记探针(在实验室内合成)进行检测(Femino et al。,1998)。 Arjun Raj教授通过使用大量较短的单标记寡核苷酸(20-mer)改进了该方法(Raj et al。,2008),该寡核苷酸整合了RNA的整个长度。这些协议可在各自的实验室网站上找到(例如,歌手实验室 href =“https://sites.google.com/site/singlemoleculernafish/home”target =“_ blank”> Raj lab )。然而,这些方案已经过时(例如,关于试剂和探针类型),并且缺乏细节。已发表了针对smFISH的方法论文,但令人惊讶的是,只有少数贴壁细胞(例如,Lee et al。,2016)。此外,许多使用smFISH的实验室经常开发用于smFISH分析的内部软件。对于缺乏编程背景的生物学家来说,这是低效的,令人困惑的,并且不是非常用户友好。

该协议最初是在Singer实验室开发的(例如,Haimovich et al。,2017),此处提供了在Gerst实验室进行的微小修改。它部分基于Raj协议和Stellaris ® RNA FISH协议(参见 Biosearch技术网站)。与其他协议的主要区别在于我们建议使用FISH-quant程序(Mueller et al。,2013; Tsanov et al。,2016),这是用户 - 友好,并希望它将用于标准化smFISH分析。


图1.描绘smFISH主要原理的方案:多个荧光标记的探针平铺mRNA的长度

关键字:mRNA, 转录, 荧光原位杂交, 单分子分辨率, 荧光显微技术, 贴壁细胞

材料和试剂

  1. 移液器吸头
  2. 显微镜玻璃载玻片25 x 75 mm x 1 mm厚(例如,Thermo Scientific,目录号:421-004T或同等产品)
  3. 玻璃盖玻片,圆形,18 mm,#1(例如,Thermo Scientific,目录号:11709875或同等产品)
  4. 1.7毫升塑料管
  5. 15毫升塑料管
  6. 无核酸酶屏障提示(10μl,200μl,1,000μl)
  7. 杂交室(例如,封闭的塑料盒,15厘米的组织培养皿,培养皿)
  8. Parafilm(Bemis,目录号:PM996)
  9. Kimwipes(例如,KCWW,Kimberly-Clark,目录号:34120或同等学历)
  10. 12孔板(例如,Costar,目录号:3513或同等产品)
  11. 铝箔
  12. 感兴趣的贴壁细胞(例如,小鼠胚胎成纤维细胞[MEF],胃癌NCI-N87细胞)
  13. 合适的培养基和补充剂(例如,补充有10%FBS和青霉素/链霉抗生物素蛋白的DMEM)
  14. (可选)细胞外基质底物,例如,纤维连接蛋白(Sigma-Aldrich,目录号:F1141-5mg)
  15. 70%乙醇
  16. 无菌PBS x1 pH 7.4,无钙,无镁(例如,Thermo Fisher Scientific,Gibco TM ,目录号:10010-015或同等产品)
  17. 10x PBS,无钙,无镁(例如,Thermo Fisher Scientific,Gibco TM ,目录号:14200-067或同等产品)
  18. MgCl 2 (例如,Sigma-Aldrich,目录号:M8266-100G或等同物)
  19. 甘氨酸(例如,Sigma-Aldrich,目录号:G8898-500G或同等物)
  20. 32%多聚甲醛(PFA)(电子显微镜科学)
  21. Surfact-Amps TM X-100(Triton X-100)10%溶液(Thermo Scientific,目录号:28314)
    注意:这种高纯度的Triton X-100效果最佳,但其他Triton X-100产品将提供令人满意的效果。
  22. 20x盐水 - 柠檬酸钠(SSC)缓冲液(例如,Sigma-Aldrich,目录号:S6639-1L或同等物)
  23. Formamide(Sigma-Aldrich,目录号:47671-250ml或同等产品)(保持在4°C)
  24. 硫酸葡聚糖(Sigma-Aldrich,目录号:D6001或同等学历)
  25. 电子。大肠杆菌 tRNA(100 mg)(Roche,目录号:10109541001)(保持在-20°C)
  26. 牛血清白蛋白(BSA)(20 mg / ml)(罗氏,目录号:10711454001)(保持在-20°C)
  27. 钒核糖核苷复合物(VRC)200 mM(例如,Sigma-Aldrich,目录号:94742-1 ml或等同物)(保持在-20°C)
  28. 不含核酸酶的水
  29. DAPI(核染色)(例如,Sigma-Aldrich,目录号:D9542-1mg或等同物)
  30. 荧光寡核苷酸探针组(例如,针对人HER2-Quasar570的Stellaris探针(Biosearch技术,DesignReady目录号:VSMF-2102-5)(参见程序A设计和生产探针)(保持 - 20°C)
  31. 抗褪色试剂(例如,来自Thermo scientific的ProLong抗褪色系列)
  32. (可选)高品质指甲油(例如,Electron Microscopy Sciences,目录号:72180)
  33. 浸油1.518,适用于显微镜/物镜
  34. PBSM缓冲区(参见食谱)
  35. 固定缓冲液(见食谱)
  36. 淬火缓冲液(见食谱)
  37. 透化缓冲液(见食谱)
  38. 预杂交(Pre-hyb)缓冲液(参见食谱)
  39. 杂交缓冲液(见食谱)(保持在-20°C)
  40. 杂交室(见食谱)
  41. DAPI染色溶液(见食谱)(保持在4°C)

设备

  1. 移液器(推荐:S1移液器,Thermo Fisher Scientific,目录号:9501)
  2. 镊子,直的,尖的,不锈钢尖端(例如,Ideal-Tek,目录号:4 SA或等效物)
  3. (可选)真空疏水阀&nbsp;
  4. 化学(油烟)罩
  5. 生物罩/生物安全柜(用于细胞培养工作)
  6. 适合您选择的细胞培养的细胞培养箱(例如,37°C,5%CO 2 )
  7. 37°C培养箱(例如,用于培养细菌板的培养箱)
  8. 用于载玻片的纸板托盘(例如,Thermo Fisher Scientific,目录号:12-587-10)
  9. 宽视场荧光显微镜(例如,Olympus,型号:BX-61;尼康,型号:Eclipse Ti-E倒置荧光显微镜或Zeiss,型号:AxioObserver Z1)配备以下产品:
    1. 荧光光源[例如,Illuminator HXP 120 V光源(Carl Zeiss,型号:Illuminator HXP 120 V)或X-cite 120 PC灯(Excelitas Technologies,X-Cite ® 120PC)]
    2. 适用于荧光团的过滤器组件+ DAPI(蓝色)过滤器
    3. 自动电动平台,用于 X , Y 和 Z 轴的亚微米运动[例如,MS 2000 XYZ 自动化阶段(ASI,型号:MS 2000)或电动 XYZ 扫描阶段,130x100 PIEZO(蔡司,产品目录号:432027-9001-000)]
    4. Plan-Apo 100x(首选)或63x油浸物镜,具有高NA(1.35 NA或更高)
    5. CCD或sCMOS高分辨率数码相机[例如,Flash 4 sCMOS(Hamamatsu)或Pixis 1024 CCD相机(Photometrics)]
    6. 适用于控制显微镜(根据制造商)的软件,用于多个通道的自动成像,多个 z - 堆叠和多个区域(例如,MetaMorph,ZEN2,μmanager)
  10. 能够进行图像处理的计算机(强大的CPU,至少32 GB RAM)
  11. 数据存储计算机
    计算机或外部驱动器上的数据存储,允许存储10 GB和最多TB的累积图像数据。

软件

  1. MATLAB-R2015a版本或更高版本&nbsp;
  2. FISH-quant(Mueller et al。,2013; Tsanov et al。,2016)(自由软件 https://bitbucket.org/muellerflorian/fish_quant )
  3. ImageJ / FIJI(Schindelin et al。,2012)(免费软件 https://imagej.net/斐济)
  4. Stellaris FISH探针设计师( https://www.biosearchtech.com/支持/工具/设计软件/ Stellaris的探头设计师);需要用户帐户(免费)&nbsp;
  5. Excel或同等程序

程序

  1. 寡核苷酸探针的设计和标记
    1. 探针是18-22聚体DNA寡核苷酸,其在一端或两端用荧光有机染料荧光标记。最常用的染料是花青(Cy),Alexa和Atto染料系列。为了设计探针,首先获得目的RNA序列。
      注意:为了获得良好的信噪比(SNR),可以检测背景中的FISH斑点,建议每个转录本使用至少25个探针(最佳= ~48个探针),这意味着短转录本(即,<~500nt)可能不适合这种版本的smFISH。替代方法如smiFISH(Tsanov等,2016),RNAscope(Wang等,2012)或clampFISH(Rouhanifard等,2018; preprint),其增强FISH信号,可能更适合短RNA。 &NBSP;
    2. 我们建议使用Stellaris探针设计网络工具。插入有义链序列并选择所需参数(即,生物体,特异性水平,探针数,探针长度和最小间距)。我们建议选择最高水平的特异性,即,5和48个20 nt探针,默认为2 nt间距。如果有&lt; 25个探头,参数可以更改,直到您满意为止。
    3. 标签探针有多种协议。最简单的解决方案,我们建议一致性和易用性,是从Biosearch技术订购Stellaris RNA FISH探针( https:// www.biosearchtech.com/ )。但是,这些都相对昂贵。一种替代方案是用胺末端命令DNA寡核苷酸并用胺活性染料标记(Singer,1998)。 请注意,此协议适用于50 nt寡核苷酸,但可用于20 nt寡核苷酸。此方法效果很好,但仍然相对昂贵。更便宜的选择是酶标记探针(Gáspár et al。,2017和2018)。&nbsp;
    4. 寡核苷酸探针组应悬浮在无核酸酶的水中(对于Stellaris探针,制备25μM溶液)并在-20℃避光保存。探针可分为10-20μl的等分试样,以避免多次冻融循环(尽管根据我们的经验,我们在多次冻融循环后未检测到任何明显的变质)。

  2. 细胞培养
    一般评论:&nbsp;
    1. 在生物安全柜(生物罩)中进行无菌细胞培养工作。
    2. 此协议专为贴壁细胞而设计。对于非贴壁细胞,需要添加额外的步骤以将细胞粘附到盖玻片上。规划此类步骤的用户需要考虑以下几点:&nbsp;
      1. 如何将细胞粘附在玻璃上(例如,通过聚赖氨酸涂层)?&nbsp;
      2. 决定在固定之前还是在最后的洗涤步骤之后将细胞粘附到玻璃上。每种情况都需要不同的方案步骤(例如,要么在盖玻片上或在管中进行洗涤,要么在坚持之前或之后进行固定),甚至可能产生不同的结果。&nbsp;
      3. 校准每个盖玻片的细胞数。&nbsp;
    3. 如果可能,建议添加额外的细胞系作为所用FISH探针的阴性对照(例如,敲除细胞,表达目标RNA的不同物种的细胞,但核苷酸序列较低同源性等)。这对于校准特定探针的FISH信号以及FISH斑点分析都很有帮助。优选证实敲除细胞系不表达目的RNA。我们注意到截短的RNA可能是从敲除细胞中表达的,这些可以通过FISH检测到。

    1. 预热1x PBS和培养基至37°C。
    2. (可选)如果涂覆盖玻片,准备涂层溶液(例如,在1x PBS中稀释纤维连接蛋白1:100)。
    3. 对于每个样品,将盖玻片放入12孔板的孔中。
    4. 用1ml 70%乙醇短暂洗涤。吸出乙醇。
    5. 用1ml无菌1x PBS短暂洗涤。
    6. (任选)加入1ml涂层溶液并根据需要孵育(例如,对于纤连蛋白,在细胞培养箱中孵育10-20分钟)。除去涂层溶液并用1ml PBS(x1)洗涤。
    7. 每孔放置1ml培养基。
    8. 选择种子贴壁细胞。&nbsp;
    9. 在细胞培养箱中培养细胞所需的实验时间;不要让文化变得融洽。固定时最大约80%-90%汇合。

  3. 鱼类
    一般评论:
    1. 为避免RNase污染样品,戴上手套,使用屏障尖端,避免在经常使用RNase的表面上工作(例如,从质粒制备试剂盒中)。&nbsp;
    2. 为安全起见,使用PFA和甲酰胺溶液的工作应在化学(烟雾)罩中进行。
    3. 除了“杂交”之外的所有步骤都在盖玻片留在孔中时进行,每孔加入1ml溶液。
    4. 建议将液体吸移到井壁上而不是直接吸附到细胞上。对于具有精细结构的细胞(例如,树突,膜纳米管),建议在最慢的设置下使用移液器辅助装置,而不是使用真空吸气器。
    5. 在洗涤步骤中无需摇动12孔板。

    1. 用PBSM洗涤细胞(3次快速冲洗)。
    2. 通过在室温(RT)下用固定缓冲液(新鲜制备)孵育10分钟(不再参见注释1)来固定细胞。
    3. 用猝灭缓冲液洗涤,室温下10分钟。&nbsp;
    4. 在室温下用PBSM洗涤10分钟。重复此步骤。此时细胞可在4℃下放置过夜。
    5. 通过与透化缓冲液在室温下孵育10分钟(不再参见注释1)来使细胞透化。
    6. 在室温下用PBSM洗涤10分钟。重复此步骤。
    7. 在室温下与Pre-hyb缓冲液孵育30分钟。
    8. 在等待(步骤C7)的同时,将预制的杂交缓冲液与探针混合,并制备杂交室(参见配方和注释2)。
    9. 将45μl杂交缓冲液置于杂交室中的每个预期的盖玻片位置。没有必要去除大气泡,但避免小泡沫状气泡。
    10. 使用镊子,轻轻地从井中取出每个盖玻片,通过触摸Kimwipe的边缘去除多余的液体,并将盖玻片的细胞面朝下放在杂交缓冲液上(参见视频1和注释3)。


      视频1.将盖玻片从12孔板转移到杂交室

    11. 用Parafilm密封杂交室,用铝箔包裹,并置于37℃培养箱中3小时至过夜。
    12. 用Pre-hyb缓冲液制备新的(或相同的)12孔板。
    13. 使用镊子将盖玻片转移回12孔板,细胞朝上。
    14. 用铝箔盖住平板,在37°C培养箱中孵育15分钟。
    15. 在Pre-hyb缓冲液中再次洗涤,在37℃下15分钟。&nbsp;
    16. 在室温下用2x SSC快速冲洗(3次快速冲洗)。
    17. 在室温下在DAPI染色溶液中染色(预热至室温)1分钟。
    18. 用2x SSC洗涤5分钟。
    19. 在最后的清洗步骤中,在纸板托盘中准备显微镜载玻片:&nbsp;
      1. 标记幻灯片。
      2. 在提起盖玻片之前,为每个盖玻片添加20μlPro-Long防褪色溶液(每个载玻片可以有两个)。去除任何气泡。另见注4。
    20. 使用镊子提起盖玻片,去除多余的液体,将细胞面朝下放在Pro-Long防褪色滴上。&nbsp;
    21. 在室温下在黑暗中干燥至少几个小时(为了获得最佳图像,请等待> 24小时)。&nbsp;
    22. (可选)对于长期储存,在Pro-Long干燥后,在盖玻片边缘周围涂指甲油。&nbsp;
    23. 幻灯片可以在RT存储几天(至少,我们没有检查超过一周)。保持在-20°C长期储存(数月至数年)。&nbsp;

  4. 成像
    重要提示:同一实验的所有幻灯片都应使用相同的条件进行成像。
    1. 可以在任何宽视场显微镜上进行成像,详见设备部分。
    2. 不要使用共焦显微镜进行smFISH成像。激光的较高光强度会导致FISH信号的快速漂白。由于smFISH信号相对较弱并且需要较长的曝光时间来收集足够的光,因此光漂白限制了所收集的光的总量。在拍摄多个 z 部分以创建3D图像堆栈时,这尤其成问题。&nbsp;
    3. 图像应在相关通道上以降序颜色[例如,Cy5(670 nm,远红色),Cy3(570 nm,黄橙色),Alexa488(520,绿色)和DAPI(460nm,蓝色)]。这在使用DAPI成像时尤其重要,因为我们已经注意到在一些细胞类型(例如,MEF,HEK293T细胞)中,通过DAPI通道成像可能导致在其他通道中出现颗粒状自发荧光。这种情况并不常见(即,每次都不会发生)但是使用几台显微镜进行复制。
    4. 曝光时间:对于FISH,建议每个FISH通道使用最大(100%)光源功率和1-3秒的曝光时间。对于DAPI,请使用低功率设置和非常短的曝光时间(例如,30%功率,20-40 ms)。
    5. 对于FISH斑点检测,更多的光子(即,更长的曝光时间)意味着更好的检测,但也增加了背景荧光。因此,用户应相应地调整时间和曝光参数。
    6. Z - 切片:为了检测整个细胞体积中的RNA斑点,应收集多个 z - 切片。建议使用0.2-0.3μm的步骤并收集至少30个切片(即,对于扁平细胞,如成纤维细胞)或更多(例如,用于HEK293T或N87细胞我们使用41 z -sections)。成像时,在切换到下一个通道之前,选择在一个通道中对所有 z - 部分进行成像的选项。
    7. 分箱:为了获得更好的分辨率,使用像素分级1 x 1.但是,有时为了有利于信号增强而不是分辨率,可以使用2 x 2分级。
    8. 有关FISH图像和 z -stacks的示例,请参见图2和视频2-5。


      图2.通过FISH-quant生成的未过滤和过滤的FISH图像的示例。 使用针对人HER2 mRNA的Stellaris探针对人NCI-N87胃癌细胞(顶行)和永生化MEF(底行)进行smFISH。在配备有Illuminator HXP 120 V光源,PlanApo 100x 1.4 NA油浸物镜和Hamamatsu Flash 4 sCMOS相机的Zeiss AxioObserver Z1 DuoLink双相机成像系统上进行成像。对于两种细胞类型,使用电动 XYZ 扫描阶段130×100PIEZO和ZEN2软件以0.0645μm/像素拍摄41步0.2μm z - 堆叠图像。图像显示中间 z - 部分的最大投影。注意,与N87相比,MEFs FISH图像(左下)的亮度增加,以允许更好地观察细胞。有关每个图像的完整 z - 堆叠,请参阅视频2-5。比例尺=10μm。


      视频2.使用人类HER-Q570探针的N87细胞的3D FISH图像


      视频3.视频2中单元格的3D滤波图像


      视频4.使用人HER-Q570探针的MEF细胞的3D FISH图像


      视频5.视频4中的单元的3D滤波图像

数据分析

FISH斑点分析由使用3D高斯拟合算法拟合衍射限制斑点的程序执行。许多实验室都会生成自己的软件/脚本。我们建议使用FISH-quant(Mueller et al。,2013; Tsanov et al。,2016),因为它是用户友好的并且可以进行多种分析( ie ,成熟mRNA,转录位点分析,共定位)。 FISH-quant的图像需要采用TIFF格式,作为每个通道获得的多个 z 部分的单独文件。如果显微镜不以TIFF格式保存文件,请使用其他程序(例如,FIJI)转换图像。按照FISH-quant的说明进行分析。
简述:

  1. 安装MATLAB,然后安装FISH-quant。
  2. 在FISH-quant的主界面中:
    1. 选择文件夹(主文件夹,图像文件夹,轮廓文件夹,结果文件夹)。
    2. 插入实验参数: XY 像素大小(基于相机和分档), Z 像素大小,油的折射率,物镜的NA,激发和发射波长FISH探针荧光团。&nbsp;
    3. 在工具→轮廓设计器下:绘制细胞,细胞核和转录位点(TS)的轮廓(提示:分别使用基于DAPI和TS / FISH染色的自动“检测细胞核”和“TS自动检测”)。&nbsp;
    4. 上传图片轮廓。
    5. 过滤背景(尝试不同的模式和参数以获得最佳过滤)。有关过滤后的图像和 z - 堆栈的示例,请参见图2和视频3和5。
    6. 根据说明执行预检测。
    7. 使用高斯算法拟合斑点。
    8. 使用阈值参数可以获得最佳效果。
    9. 保存检测设置。
  3. 从工具→批处理可以执行多个图像的分析。批量分析后可以修改阈值参数以微调结果。建议设置参数,使阴性对照接近0个点。&nbsp;
  4. 使用工具→点检查器消除明显的误报(例如,高自发荧光区域)。&nbsp;
  5. 来自FISH-quant的数据保存为'.txt'文件。可以将数据复制并粘贴到Excel中进行进一步分析(例如,图3)。


    图3.人类N87癌细胞和MEF中人HER2 mRNA表达水平的FISH-quant分析。每个圆/三角形代表单个细胞的斑点数。

笔记

  1. 要保持的最关键的孵育时间是固定和透化步骤。对于所有其他洗涤步骤,样品可以保留更长的孵育时间。根据我们的经验,淬火/洗涤30分钟和Pre-hyb长达60分钟对FISH没有任何不利影响。较短的孵育时间(例如,5分钟)或较少的洗涤次数(例如,一次而不是固定/透化后两次)具有小的但不同的不利影响。缩短杂交后洗涤时间将导致背景信号的显着增加。该方案中的孵育时间适用于许多细胞类型(包括MEF,许多癌细胞系,小鼠原代海马神经元等)。然而,孵育时间可能需要用户优化以适合他们自己的细胞系。
  2. 使用荧光团时无需调暗灯光,但建议在添加抗褪色溶液之前尽量减少光照。在成像过程中房间应该变暗,以尽量减少自发荧光并避免背景光污染。&nbsp;
  3. 使用镊子轻轻用力抓住并抬起盖玻片,因为盖玻片很容易折断。如果盖玻片破裂成两个大块,它仍然可以挽救,并且实验可以继续(确保将两个碎片分开,这样它们就不会将一个碎片粘在另一个上面)。此外,如果镊子没有牢牢地握住盖玻片,盖玻片可能会掉到工作台上。同样,盖玻片仍然可以挽救,但是用户需要猜测细胞的正确侧面。
  4. 还有其他商业上可获得的抗褪色溶液,以及可以使用的实验室制备的抗褪色溶液(例如,通过使用葡萄糖氧化酶)。但是,我们没有经验,这些可能不适合FISH或长期储存。

食谱

  1. PBSM缓冲液(500毫升)
    50毫升10倍PBS
    2.5毫升MgCl 2 1M
    475毫升无核酸酶水
    在RT存储
  2. 固定缓冲液(8毫升)
    7毫升PBSM
    1毫升PFA 32%
    准备新鲜
  3. 淬火缓冲液(10毫升)
    9.5毫升PBSM
    0.5毫升2 M甘氨酸
    准备新鲜,因为它往往会得到微生物污染
    提前准备库存解决方案:&nbsp;
    1. PBSM缓冲液(食谱1)
    2. 2 M甘氨酸原液在水中,过滤灭菌(在室温下储存)
  4. 透化缓冲液(100毫升)
    10毫升10倍PBS
    1ml Surfact-Amps TM X-100 10%
    89毫升无核酸酶水
    在RT存储
  5. 预杂交(Pre-hyb)缓冲液(10 ml)
    1毫升20倍SSC
    1毫升甲酰胺(使用前温热至室温)
    8毫升无核酸酶水
    准备新鲜
  6. 杂交缓冲区&nbsp;

    向杂交缓冲液中添加新鲜探针。建议首次使用探头时校准探针浓度(默认值:250 nM或10 ng /样品)。

    提前准备库存解决方案:
    1)10mg / ml E.大肠杆菌 tRNA溶液在无核酸酶的水中。储存在-20°C。
    2)20%硫酸葡聚糖水溶液(粘稠,需要30-60分钟才能完全溶解)。在RT存储。
  7. 杂交室
    1. 塑料盒或塑料盘(例如,培养皿,15cm组织培养皿)。腔室的尺寸应适应所有盖玻片,允许盖玻片之间至少几毫米
    2. 将封口膜放在室底部。避免盖玻片放置的皱纹
    3. (可选)绘制网格(每个正方形应适合单个盖玻片)并标记正方形
    4. 取15毫升锥形管的盖子,将其放在室的边缘。用1毫升水或缓冲液填充盖子(这需要保持腔室内的湿度)
    5. 放置盖玻片后,准备一块封口膜以密封腔室
    6. 准备铝箔以覆盖腔室以防止光线照射
    7. 塑料腔可以多次重复使用
  8. DAPI染色液(200毫升)
    20毫升20倍SSC
    10μl10mg/ ml DAPI(终浓度:0.5μg/ ml)
    180毫升无核酸酶水
    在4°C的黑暗中储存
    提前准备DAPI股票解决方案:
    10mg / ml DAPI水溶液。在-20°C的黑暗中储存

致谢

G.H.是Koshland基金会和McDonald-Leapman Grant高级博士后奖学金的获得者。这项工作的资金来自Joel和Mady Dukler癌症研究基金(WIS)的赠款,Moross综合癌症中心,Weizmann研究所和美国以色列国家科学基金会 - 国家科学基金会的原则证明(# 2015846)到JEG

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Buxbaum,A.R.,Haimovich,G。和Singer,R。H.(2015)。 在正确的时间在正确的位置:可视化和了解mRNA定位。 > Nat Rev Mol Cell Biol 16(2):95-109。
  2. Buxbaum A. R.,Wu B.和Singer R. H.(2014)。 神经元中的单个β-肌动蛋白mRNA检测揭示了调节其可译性的机制。 Science 343(6169):419-422。&nbsp;
  3. Cabili,M.N.,Dunagin,M.C.,McClanahan,P.D.,Biaesch,A.,Padovan-Merhar,O.,Regev,A.,Rinn,J.L。和Raj,A。(2015)。 单细胞和单分子分辨率下人类lncRNA的定位和丰度分析。 Genome Biol 16:20。
  4. Chou,Y.Y.,Heaton,N.S。,Gao,Q.,Palese,P.,Singer,R。H. and Lionnet,T。(2013)。 病毒出芽前细胞质中不同流感病毒RNA片段的共定位,如单分子灵敏度FISH所示分析。 PLoS Pathog 9(5):e1003358。
  5. Femino,A.M.,Fay,F。S.,Fogarty,K。和Singer,R。H.(1998)。 单个RNA转录本的可视化原位。 >科学 280(5363):585-590。
  6. Gáspár,I.,Wippich,F。和Ephrussi,A。(2017)。 酶法生产单分子FISH和RNA捕获探针。 RNA < / em> 23(10):1582-1591。
  7. Gáspár,I.,Wippich,F。和Ephrussi,A。(2018)。 末端脱氧核苷酸转移酶介导标记探针的产生,用于单分子FISH或RNA捕获。 生物方案 8(5):e2750。
  8. Haimovich,G.,Ecker,C.M.,Dunagin,M.C.,Eggan,E.,Raj,A.,Gerst,J.E。和Singer,R.H。(2017)。 通过哺乳动物细胞中膜纳米管延伸的细胞间mRNA运输。 Proc Natl Acad Sci USA 114(46):E9873-E9882。
  9. Lee,C.,Roberts,S.E。和Gladfelter,A。S.(2016)。 使用单分子FISH对多核细胞中转录物进行定量空间分析。 方法 98:124-133。
  10. Mueller,F.,Senecal,A.,Tantale,K.,Marie-Nelly,H.,Ly,N.,Collin,O.,Basyuk,E.,Bertrand,E.,Darzacq,X。和Zimmer,C 。(2013)。 FISH-quant:自动计算3D FISH图像中的成绩单。 Nat方法 10(4):277-278。
  11. Pichon,X.,Lagha,M.,Mueller,F。和Bertrand,E。(2018)。 一个不断发展的工具箱,用于在单个细胞中对基因表达进行成像:针对严苛挑战的敏感方法。 Mol Cell 71(3):468-480。
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引用:Haimovich, G. and Gerst, J. E. (2018). Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells. Bio-protocol 8(21): e3070. DOI: 10.21769/BioProtoc.3070.
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Gal Haimovich
Molecular Genetics, Weizmann Institute of Science, Israel
In the following link you can find a protocol for FISH-IF (smFISH-immunofluorescence), which is largely based on the above protocol: https://bio-protocol.org/MyLab.aspx?id=3013&labid=81
2018/11/4 22:13:58 Reply
Gal Haimovich
Molecular Genetics, Weizmann Institute of Science, Israel

Sorry, the direct link is: https://bio-protocol.org/p631

2018/11/4 22:16:03