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Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture
末端脱氧核苷酸转移酶介导的用于单分子FISH或RNA捕获的标记探针的生成   

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RNA
Oct 2017

Abstract

Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

Keywords: Terminal deoxynucleotidyl transferase (末端脱氧核苷酸转移酶), Labelled terminator nucleotide (标记的终止子核苷酸), Probe production (探针生成), Single molecule FISH (smFISH) (单分子FISH(smFISH)), RNA capture (RNA捕获), RNA affinity purification (RAP) (RNA亲和纯化(RAP))

Background

The use of multiple, singly-labeled, short oligonucleotides of synthetic origin has vastly improved the detection of specific transcripts with high specificity and single molecule sensitivity (Femino et al., 1998; Raj et al., 2008). Such probe molecules have improved penetration and require milder hybridization conditions than the classically used long nucleic acid probes, resulting in better preservation of the structure of the specimen (e.g., Little et al., 2015, Gaspar et al., 2017a). Since in this design multiple oligonucleotides–typically 24-96–target different portions of the same transcript, there occurs an accumulation of signal on the specific target molecules over the aspecific background, as opposed to the equal signal produced by long multiply labeled probes (Raj et al., 2008). Moreover, as the labeling of the individual short probes is quantitative–as opposed to the stochastic labelling of the long probes–the signal intensity directly and linearly correlates with the transcript copy number at a given spot, allowing precise recording/counting of the target RNA molecules (Raj et al., 2008, Little et al., 2015). Until now, the production of smFISH probe arrays has depended on chemical synthesis and labeling that rendered such single molecule FISH application inflexible and costly. Here, we describe an effective and cost-efficient enzymatic three-pot probe production (3P3) assay that makes use of terminal deoxynucleotidyl transferase (TdT) and custom labeled terminator nucleotides to convert any custom-assembled array of cheap PCR oligos into smFISH probes bearing fluorescent or non-fluorescent labels of the experimenter’s choice (Gaspar et al., 2017b). These enzymatically produced 3P3 probes are chemically nearly identical to smFISH probes from other sources. Thus the same protocols–optimized for a given specimen under study–can be used to perform single molecule FISH (reviewed in Gaspar and Ephrussi, 2015) and RNA capture analyses(see e.g., Gaspar et al., 2017a and Khong et al., 2017).

Materials and Reagents

  1. 1.5 ml Eppendorf tube (e.g., Sigma-Aldrich, catalog number: Z336769 )
  2. 0.2 ml thin-walled PCR tube (e.g., Corning, catalog number: 6571 )
  3. 2 cm thick adhesive tape (Tesa)
  4. Glass slides (e.g., VWR, catalog number: 631-0411 ) and coverslips (e.g., 22 x 22 x 0.17 mm, Marienfeld-Superior, catalog number: 0107052 ) for sample preparation
  5. 15 ml tubes (e.g., Corning, Falcon®, catalog number: 352097 )
  6. 0.22 μm filter (e.g., Corning, catalog number: 431227 )
  7. 3 cm wide foldback paperclips (e.g., Staples, catalog number: WW-9130156 )
  8. Amine reactive labels (tested and working):
    1. BDP-FL-NHS (Lumiprobe, catalog number: 11420 )
    2. Atto-tec Atto488-NHS (Atto-tec, catalog number: AD 488-31 ), Atto532-NHS (Atto-tec, catalog number: AD 532-31 ), Atto565-NHS (Atto-tec, catalog number: AD 565-31 ) and Atto633-NHS (Atto-tec, catalog number: AD 633-31 )
    3. AlexaFluor488-NHS (Thermo Fisher Scientific, InvitrogenTM, catalog number: A20000 )
    4. Abberior STAR 470SXP-NHS (Abberior, catalog number: 1-0101-008-3 ) and Abberior STAR RED-NHS (Abberior, catalog number: 1-0101-011-3 )
    5. biotin-NHS (Sigma-Aldrich, catalog number: H1759 )
  9. Anhydrous DMSO (e.g., Sigma-Aldrich, catalog number: 276855 )
  10. Optional: silica gel (e.g., Merck, catalog number: 1.01969.1000 ) (see Note 1)
  11. Amino-11-ddUTP (Lumiprobe, catalog number: 15040 ) or 5-propargylamino-ddUTP (Jena Biosciences, catalog number: NU-1619 )
  12. 1 M NaHCO3, pH 8.4 (e.g., Sigma-Aldrich, catalog number: S5761 )
  13. A custom designed target specific array of non-overlapping ssDNA oligonucleotides (desalting purification is sufficient, see Software section for the design)
  14. 20 U/μl Terminal deoxynucleotidyl transferase (TdT) with 5x TdT buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0161 )
  15. 1-3 M Na-acetate, pH 5.5 (e.g., Sigma-Aldrich, catalog number: S2889 )
  16. 5 mg/ml linear acrylamide (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9520 )
  17. Ethanol (e.g., Merck, EMD Millipore, catalog number: 1.00983 )
    1. 100% ethanol, -20 °C
    2. 80% ethanol, 4 °C
    3. 70% ethanol, RT
  18. Nuclease free ddH2O (e.g., New England Biolabs, catalog number: B1500S )
  19. 40% Acrylamide/Bis solution, 29:1 (e.g., Bio-Rad Laboratories, catalog number: 1610146 )
  20. Urea (e.g., Sigma-Aldrich, catalog number: U5378 )
  21. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (e.g., Sigma-Aldrich, catalog number: T9281 )
  22. 10% (w/v) ammonium persulfate (APS) (e.g., Sigma-Aldrich, catalog number: A3678 )
  23. 6x gel loading dye (e.g., New England Biolabs, catalog number: B7021S )
  24. SYBR-GOLD (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494 )
  25. Optional: colorimetric Biotin Assay Kit (e.g., Sigma-Aldrich, catalog number: MAK171 ) (see Note 8)
  26. 20 mg/ml Proteinase-K (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2546 )
  27. Mounting medium
    1. VectaShield (Vector Laboratories, catalog number: H-1000 )
    2. 80% TDE (see Recipes)
  28. Pierce® Avidin agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20219 )
  29. Dynabeads® MyOneTM C1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 65001 )
  30. Quick-RNATM MicroPrep Kit (Zymo Research, catalog number: R1050 )
  31. Tris-HCl pH 7.0 (e.g., Sigma-Aldrich, Roche Diagnostics, catalog number: 10812846001 )
  32. Colorimetric Biotin Assay Kit (Sigma-Aldrich, catalog number: MAK171 )
  33. Tris base (e.g., Sigma-Aldrich, catalog number: T1503 )
  34. Ethylenediaminetetraacetic acid (EDTA) (e.g., Sigma-Aldrich, catalog number: E5391 )
  35. Sodium chloride (NaCl) (e.g., Merck, catalog number: 106404 )
  36. Potassium chloride (KCl) (e.g., Merck, catalog number: 104936 )
  37. Potassium dihydrogen phosphate dihydrate (KH2PO4·2H2O) (e.g., Merck, catalog number: 104873 )
  38. Sodium phosphate dibasic (Na2HPO4) (e.g., Merck, catalog number: 106342 )
  39. EM-grade paraformaldehyde (e.g., Electron Microscopy Sciences, catalog number: 15710 )
  40. Triton X-100 (e.g., Sigma-Aldrich, catalog number: X100 )
  41. Boric acid (e.g., Merck, catalog number: 100165 )
  42. Ethylene carbonate (e.g., Sigma-Aldrich, catalog number: E26258 )
  43. 50 mg/ml heparin (e.g., Sigma-Aldrich, catalog number: H3393 )
  44. 10 mg/ml salmon sperm DNA (e.g., Sigma-Aldrich, catalog number: D7656 )
  45. 2,2’-Thiodiethanol (Sigma-Aldrich, catalog number: 166782 )
  46. 20% (v/v) SDS (e.g., Sigma-Aldrich, catalog number: 05030 )
  47. PMSF (e.g., Sigma-Aldrich, catalog number: P7626 )
  48. cOmplete® mini EDTA-free protease inhibitor (Roche Diagnostics, catalog number: 11836170001 )
  49. RiboLock RNase Inhibitor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
  50. Sodium citrate (e.g., Sigma-Aldrich, catalog number: S1804 )
  51. TE buffer (see Recipes)
  52. 1x PBS (see Recipes)
  53. Fixative (see Recipes)
  54. PBT (see Recipes)
  55. 1.5x PAGE loading buffer (see Recipes)
  56. 1x and 10x TBE (see Recipes)
  57. 15% PA - 8 M Urea stock (see Recipes)
  58. 20x SSC buffer (see Recipes)
  59. 2x full-HYBEC (see Recipes)
  60. 2x wash-HYBEC (see Recipes)
  61. Lysis buffer (see Recipes)
  62. Capturing hybridization buffer (see Recipes)
  63. Low salt wash buffer (see Recipes)
  64. High salt wash buffer (see Recipes)
  65. Elution buffer (see Recipes)

Equipment

  1. Optional: inert gas (e.g., Argon) glove-box (e.g., Inert Technology, model: PureLab HE 2GB ) (see Note 1)
  2. PCR machine with programmable hot-lid (e.g., Bio-Rad Laboratories, catalog number: 1851148 )
  3. -20 °C freezer
  4. Refrigerated table-top centrifuge (e.g., Eppendorf, catalog number: 5426000018 )
  5. Erlenmeyer flask
  6. Handcast PAGE system including a 1 mm spacer plate (e.g., Bio-Rad Laboratories, catalog number: 1653311 ), a short plate (e.g., Bio-Rad Laboratories, catalog number: 1653308 ) and a 15-well comb (e.g., Bio-Rad Laboratories, catalog number: 4560016 )
  7. Vertical Electrophoresis Cell (e.g., Bio-Rad Laboratories, catalog number: 1658005 )
  8. Electrophoresis power supply (e.g., Bio-Rad Laboratories, catalog number: 1645050 )
  9. Gel documentation system with filters to image fluorescence of SYBR-GOLD and the fluorescent dye used for labeling (e.g., Bio-Rad Laboratories, catalog number: 17001402 )
  10. P2, P200 and P1000 pipettes
  11. Rocking thermoblock (e.g., Eppendorf, model: ThermoMixer® C , catalog number: 5382000015)
  12. Microscope for imaging (we use a Leica SP8 (Leica, model: Leica TCS SP8 ) equipped with a 63x NA=1.4 oil immersion objective and two HyD detectors)
  13. Tissue grinder (e.g., DWK Life Sciences, Kimble, catalog numbers: 8853000015 or 8853000040 )
  14. Rotator (e.g., Cole-Parmer, Stuart, model: Rotator SB3 )
  15. Magnetic rack (e.g., New England Biolabs, catalog number: S1507S )
  16. Moisture free chamber (see Note 1)
  17. UV/VIS spectrophotometer (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 8000 , catalog number: ND-8000-GL)
  18. Nutator (e.g., Labnet International, model: S0500 )

Software

  1. A probe designer algorithm, e.g., the StellarisTM Probe Designer
    (https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer, registration required) or the provided smFISHprobe_finder.R script (Supplementary File 1, see Notes 2 and 17)
  2. MS Office Excel to run the interactive probe_calculator.xls sheet (Supplementary File 2)
  3. ImageJ/FIJI (https://imagej.nih.gov/ij/) with the xsPT plugin (https://github.com/Xaft/xs/blob/master/_xs.jar)
  4. Optional: deconvolution software, e.g., Huygens Essentials (https://svi.nl/Huygens-Essential) or DeconvolutionLab2 (Sage et al., 2017; http://bigwww.epfl.ch/deconvolution/deconvolutionlab2/)
  5. R (preferentially with RStudio) for data analysis
  6. smFISH_analysis.R to analyze the sensitivity and specificity of smFISH (Supplementary File 3)

Procedure


Figure 1. Graphical overview of the three-pot probe production assay. A. Conjugation of the label to the NH2-ddUTP terminator nucleotide; B. TdT mediated labeling of ssDNA oligonucleotides; C. Purification of labeled ssDNA molecules (probes).


  1. Preparing the dye conjugated ddUTP (First pot, Figure 1A)
    1. Reconstitute the dye-NHS ester to 40 mM final concentration in anhydrous DMSO in a moisture free environment. (see Note 1)
    2. In a clean 1.5 ml Eppendorf tube, aliquot X μl of 20 mM Amino-11-ddUTP or 5-propargylamino-ddUTP. (see Note 3)
    3. Add 0.2X μl of 1 M NaHCO3 (pH = 8.4).
    4. Finally, add X μl of 40 mM dye-NHS ester (see Note 4). Mix well and incubate at RT for 2 h sealed from light.
    5. After the incubation, add 1.8X μl dH2O to expand the reaction volume to 4X μl. This results in a 5 mM stock of dye conjugated ddUTP (see Note 5). The resulting labeled nucleotide is stable for over a year when stored at -20 °C.

  2. Production of labeled ssDNA oligonucleotides (second pot, Figure 1B)
    1. Prepare an equimolar mixture of all different ssDNA oligos that should be labeled together, i.e., that target the same transcript. The total concentration of oligos should not be lower than 100 μM. (see Note 6)
    2. To label 1 nmol of oligonucleotide mixture, prepare the following labeling mixture in a 0.2 ml thin-walled PCR tube:
      Amount
      Final concentration
      1 nmol oligo-mixture (e.g., 4 μl of 250 μM stock)
      66.67 μM
      3 μl 5x TdT reaction buffer (with Co2+)
      1x
      3-5 nmol dye-ddUTP (e.g., 0.6-1.0 μl of 5 mM stock)
      200-330 nM
      1x standard amount of TdT
      0.4-0.8 U/μl
      dH2O
      to 15 μl
      Please use the interactive probe_calculator.xls Excel sheet (Supplementary File 2) to get the reaction composition adjusted to the dye-ddUTP used for labeling.
    3. Incubate at 37 °C O/N (16-18 h) in a PCR machine with the hot-lid set to 37 °C.

  3. Purification and spectroscopic analysis of the labeled oligonucleotide mixture (third pot, Figure 1C)
    1. After the O/N incubation, add 60 μl of 1 M Na-acetate (pH = 5.5), 125 μl of dH2O and 1.5 μg linear acrylamide to the reaction mixture.
    2. Transfer the entire 200 μl into a clean 1.5 ml Eppendorf tube. Add 800 μl 100% ethanol prechilled at -20 °C. Invert the tube a couple of times and place it into the -20 °C freezer for about 15-20 min.
    3. In the meantime, cool the table-top centrifuge to 4 °C.
    4. After the -20 °C incubation, spin the oligonucleotide mixture at 16,000 x g for 20 min at 4 °C.
    5. Remove the supernatant and add 1 ml 80 % ethanol prechilled at 4 °C. Vortex until the pellet dissociates from the bottom of the tube.
    6. Spin at 16,000 x g for 5 min at 4 °C.
    7. Wash away the pellet from the wall of the tube with 1 ml 80% ethanol and transfer the entire volume including the floating pellet into a clean 1.5 ml Eppendorf tube. Repeat Steps C5 and C6 two more times.
    8. Remove the supernatant from the last wash and let the pellet dry on air. (see Note 7)
    9. Resuspend the dried pellet in 15-50 μl nuclease-free dH2O.
    10. Measure the absorbance of the labeled oligonucleotide mix at 260 nm and at the dye absorption maximum (e.g., 570 nm for Atto565).
    11. In order to calculate the concentration and the degree-of-labeling (DOL), measure the absorbance of the unlabeled, undiluted oligonucleotide mix at 260 nm. (see Note 8)
    12. Calculate the molar extinction coefficient (𝜖oligo) of the oligonucleotide mixture by dividing the measured OD260 nm value with the concentration of the mixture (in M). Increase this value by 9,000 mol-1 cm-1 to correct for the UTP added to the 3’ of the ssDNA molecules.
    13. Calculate the concentration of the labeled oligo by dividing the dye-corrected OD260 nm absorption by 𝜖oligo:coligo = (OD260 nm - cf260 nm x ODdye)/𝜖oligo (see Note 9).
    14. Calculate the concentration of the dye as follows: cdye = ODdye/𝜖dye (provided by the dye manufacturer).
    15. Typically, only a small fraction of the dye molecules is present as contaminants (i.e., free from ssDNA), therefore the DOL is estimated as follows: DOL = cdye/coligo.
    16. The fraction of recovered oligonucleotides is assessed by taking the ratio of the recovered and initial amounts of the oligo (recovery% = coligo x Vresuspension/ninitial).
    17. The measured OD values can be entered into the interactive probe_calculator.xls Excel sheet (Supplementary File 2) to obtain the concentration and the DOL of the labeled oligonucleotide mixture.
    18. Store the labeled probes at -20 °C.

  4. Trouble-shooting of 3P3 probe production
    Labeled oligo mixtures with 0.9 < DOL ≤ 1.0 are considered good quality products that can be used in smFISH applications.
    1. The most typical cause of DOLs lower than 0.9 is the composition of the oligonucleotide mixture. We currently lack an understanding of which property(s) of the mixture–e.g., formation of intra- and intermolecular hybrids in the mixture–influence the labeling efficiency. However, if DOL < 0.9 is obtained, we recommend ‘splitting’ the oligo mixture into two-three non-overlapping fractions, i.e., preparing two-three mixtures of the ssDNA oligos present in the original mixture. Individual labeling of these mixtures can help in identifying any molecules that behave extraordinarily in the labeling reaction. In most cases, we found that this ‘splitting’ almost completely alleviates the low labeling efficiency problem, i.e., the DOL of each of the split mixtures increases above 0.9.
    2. Another solution we found was to re-label the already labeled oligonucleotide mixture, i.e., start over the labeling protocol from Step B2 using the labeled ssDNA molecules as input. (REF)
    3. The second most frequent reason for low DOL is the non-accurate (lower than actual) measurement of the molar extinction coefficient of the unlabeled oligo mixture. This can be noticed that when calculating the fraction of recovered oligonucleotides (recovery %) one obtains a value higher than one. In such cases, we recommend re-measuring the OD260 nm of the unlabeled oligo mixture at different concentrations (e.g., at 50, 100 and 200 μM) and estimating the 𝜖oligo by taking the slope of the line fitted to the data points.
    4. We found that terminal transferase activity of TdT varies from batch to batch. This may result in improper labeling (DOL < 0.9) when using 1x standard amount of the enzyme. We recommend to redefine the standard amount when starting a new aliquot of TdT, by setting up several labeling reactions of a single ssDNA oligo with Atto633-ddUTP (Procedures B and C) using increasing concentrations of TdT–e.g., 1x, 2x and 3x of the standard amount found in the recipe generator. Use the smallest concentration of the enzyme that yields DOL > 0.9 as the new standard amount.
    5. DOL > 1.0 may also indicate an imprecisely determined 𝜖oligo. This would appear as a huge loss of the oligos (recovery < 50%). Try the same solution as in Step D3.
    6. If the 𝜖oligo is appropriate, DOL > 1.0 indicates free dye contamination. In such cases it is advised to measure the DOL by PAGE (Procedure E). If the DOL determined by densitometry is ~1.0 and the difference of the two DOLs is small (max. 0.05-0.1), the free dye contamination is considered harmless in the downstream applications. If it is higher, re-purification of the oligonucleotide mixture by another method (e.g., by size exclusion chromatography) is recommended. We observed such high free dye contamination when using Abberior470SX-, Atto488- and AlexaFluor488-ddUTP (Gaspar et al., 2017b).

  5. PAGE analysis of the labeled oligonucleotides
    Gel electrophoresis provides a simple means to confirm the DOL estimated from spectroscopy data and to calculate DOL in case of non-fluorescent modifications (e.g., biotinylation). PAGE analysis also allows the quality control of most of the labeled ddUTP analogs, i.e., to determine whether there are unconjugated ddUTP molecules in the dye conjugated ddUTP stock (produced in Procedure A) that will result in reduced DOL of oligonucleotides.
    IMPORTANT: Only oligo-mixtures containing ssDNA molecules of identical length (e.g., only 20 mers) should be analyzed by PAGE.
    1. Wipe the glass plates for gel casting clean with 70% ethanol, assemble the cassette and seal with 20 mm wide transparent tape, leaving the top (where the comb will be inserted) open. Put two fold-back clamps on the two sides of the cassette such that the clamps clamp above the spacer between the two glass plates.
    2. To cast a 10 x 8 cm acrylamide gel, add 8 ml of 15% PA - 8 M urea stock (see Recipes), 40 μl 10% APS and 5 μl TEMED into an Erlenmeyer flask. Mix well by swirling the flask and pour the mixture between the two plates of the assembled cassette. Fill to the top.
    3. Insert the comb, place the cassette horizontally and wait until the gel polymerizes (15-20 min).
    4. Remove the clamps, the tape and the comb. Rinse the outside of the cassette with dH2O to remove gel pieces polymerized on the outer surface and assemble the PAGE chamber.
    5. Fill with 1x TBE buffer (see Recipes) and pre-run the gel for 30 min with 2.5 mA/cm current (20 mA for an 8 cm long gel).
    6. In the meantime, prepare 5 μl of each sample by mixing 3 μl 1.5x PAGE loading buffer (see Recipes) and 2 μl oligonucleotide mixture containing 15-60 pmoles of labeled oligo. As size marker use a dilution row of the unlabeled oligonucleotide mixture (e.g., 1.5, 3 and 6 pmoles), no boiling is necessary.
    7. After the pre-run (E5), rinse all wells on the gel with 1x TBE using a P200 pipette to remove the accumulated urea that would prevent the loading.
    8. Load the samples and run the gel until the xylene cyanol (blue) and the bromophenol blue (purple) markers (from the 6x gel loading dye) reach about the one-third and two-third of the gel length.
    9. Image the fluorescently labeled pool of molecules on a gel-imager with appropriate filter sets to excite and detect the incorporated fluorescent dyes.
    10. Incubate the gel with SYBR-GOLD (or similar RNA/ssDNA dye) diluted 1:10,000 in 1x TBE for 10-15 min.
    11. Re-image the gel to detect both the non-modified and modified pools of ssDNA.
    12. Due to the addition of a bulky terminator nucleotide, labeled oligonucleotides run slower and thus they are well separated from their non-modified peers during PAGE. The amount of non-modified oligos can be measured by comparing the corresponding SYBR-GOLD fluorescence intensity to that of the dilution row of the unlabeled oligonucleotide mixture used as loading control (Figures 2A and 2A’).
    13. The presence of unconjugated ddUTP will result in production of unlabeled, ddUTP-terminated oligonucleotides. On gel, they appear as an intermediate band migrating between the unlabeled and labeled, fluorescent pool of oligos.


      Figure 2. PAGE analysis of the labeled oligonucleotide mixtures. Labelling of the osk20nt-15x (Gaspar et al., 2017b) probe mixture with Atto633-amino-11-ddUTP (lane 4) or Atto633-5-propargylamino-ddUTP (lane 5). The fluorescently labeled ssDNA species (magenta, A) migrate slower in the gel than the unlabeled oligonucleotides (green, A, gray, A’). Note that the probe molecules labeled with far-red fluorescence are not visible in the SYBR-GOLD channel, possibly because of a very efficient energy transfer form SYBR GOLD to Atto633 that quenches the SYBR GOLD fluorescence. While this is the case with other far-red dyes also (e.g., Abberior-RED), when there is no quenching dye present (e.g., biotinylated probes) or when the FRET fluorescence can be detected by the SYBR GOLD imaging setup (e.g., in case of Atto565), the labeled probes appear in the SYBR GOLD channel (Gaspar et al., 2017b). 1.5, 3, 6, 20 and 20 pmol ssDNA mixture was loaded to lanes 1-5, respectively. After a labeling reaction with either of the terminator nucleotides, only low amounts (< 1.5 pmol) of unlabeled oligonucleotides were left in the mixtures (lanes 4 and 5A and 5A’), indicating near-quantitative labeling with both terminator nucleotides. The red arrow indicates fluorescence of the bromophenol blue dye.

  6. Single molecule FISH in Drosophila ovaries
    1. Dissect ovaries into a 1.5 ml Eppendorf tube containing 300-500 μl fixative (for ovary dissection, please refer to Gaspar and Ephrussi, 2017).
    2. Fix for 20 min by nutating the dissected material.
    3. Remove the fixative and rinse the ovaries with 1 ml PBT (see Recipes).
    4. Wash ovaries in 1 ml fresh PBT for 10 min while nutating.
    5. Replace PBT and add Proteinase-K to 2 μg/ml final concentration. Nutate for 5 min at room temperature (RT). (see Note 10)
    6. Preheat 0.5 ml 0.05 v/v % SDS in PBS (see Recipes) to 95 °C.
    7. After the 5 min Proteinase-K digestion, remove the PBT and immediately apply the preheated SDS/PBS to the ovaries. Incubate them for 5 min at 95 °C.
    8. Add 1 ml RT PBS to the ovaries to cool the solution.
    9. Replace the wash solution with 200 μl 2x full-HYBEC (see Recipes).
    10. Transfer the tube to a rocking thermoblock set to 37-42 °C and shake at 1,000 RPM for 10 min.
    11. In the meantime, prepare the probe solution (5-12.5 nM/individual probe) in 50 μl of 2x full-HYBEC. Transfer the probe solution to the thermoblock and allow it to warm up (2-3 min). (see Note 11)
    12. After the 10 min incubation of the ovaries, apply the probe solution by mixing it into the 200 μl 2x full-HYBEC already on the specimen.
    13. Incubate for 1.5-3 h at 37-42 °C while rocking at 1,000 RPM.
    14. 10-15 min before the end of the incubation, prewarm two times 1 ml 2x wash-HYBEC (see Recipes) to the hybridization temperature.
    15. Remove the hybridization solution–it may be kept at 4 °C for another hybridization–and wash with 1 ml prewarmed 2x wash-HYBEC for 2 x 15 min.
    16. Wash once with 1 ml RT PBT for another 15 min.
    17. Thoroughly remove the PBT and apply mounting medium, e.g., 80% TDE (see Recipes) or VectaShield. (see Note 12)
    18. After allowing the mounting medium to soak for at least one hour, mount the ovaries onto glass slides and image them using a high NA (> 1.1 NA) objective (Figure 3A).


      Figure 3. smFISH analysis of oskar mRNA in developing Drosophila egg-chambers. A. oskar mRNA is produced in the transcriptionally active nurse cells (left) and it is transported into the oocyte, where it localizes eventually at the posterior pole (on the right). The female germ-line cells (nurse cells + oocyte) are encapsulated by a layer of somatic epithelium, the follicle cells that do not express oskar mRNA. B. Close-up of the boxed region in A. oskar mRNA was detected by two different probe sets osk37x-Atto532 (green, osk3UTR#1-15 + oskCD#1-22) and oskCD20x-633 (red, oskCD#23-42, Gaspar et al., 2017b). There is a high degree of co-localization of the two puncta-like signal in the nurse cell compartment (B) and no such strong accumulations of the probes is detected in the follicle cells (B-B”). Arrows indicate multiple transcriptional loci inside the polyploid nucleus of the nurse cell (B). Scale bars represent 10 μm. C and D. Correlation of the signal intensities of the two channels. C. When all objects across the egg chamber are considered, a strong linear correlation (R2 = 0.972) is established, indicating that the RNA content of the detected objects is variable. By fitting multiple Gaussian functions to the signal intensity distribution (Little et al., 2015; Gaspar et al., 2017b), it is possible to filter the smFISH objects containing a single copy of oskar RNA. D. In this regime, the correlation of the signal intensities is low to moderate (R2 = 0.264), however, vast majority of the objects can be detected in both channels–e.g., 88.6% of the objects analyzed in the Atto633 channel are also detectable in the Atto532 channel also. Vertical and horizontal black dashed lines represent the detection thresholds for the Atto633 and Atto532 channels, respectively. Red dashed line shows the boundary between objects with single and multiple copies of RNA (C). Colours represent the relative intensity difference between the two channels (see panel C for key). The number of smFISH objects (number of experiments) is indicated in C and D.

  7. RNA capture from ovarian lysate
    1. Isolate ovaries from well-fed 2-3 day old Drosophila melanogaster in PBS (see Note 13).
    2. Remove PBS and resuspend with 3 volumes lysis buffer.
    3. Mechanically homogenize using a tissue grinder.
    4. Transfer to fresh 15 ml tubes and clear the lysate by centrifugation (5 min at 140 x g).
    5. Transfer the supernatant to a fresh tube and dilute with 2 volumes capturing hybridization buffer (see Recipes).
    6. Preclear by adding 1:50 v/v Pierce® Avidin Agarose prewashed with a 1:2 mix of lysis buffer and capturing hybridization buffer.
    7. Keep on a rotator for 30 min at room temperature.
    8. Remove Avidin agarose by centrifugation (5 min at 140 x g).
    9. Split the precleared lysate (keep 0.1% as input sample) and supplement it with 0.25 µg (see Note 14) of targeting and non-targeting control biotinylated DNA probes per ml of ovaries and incubate at 37 °C for 2 h on a rotator.
    10. Pre-wash 3.75 µl magnetic Streptavidin beads per ml lysate with a 1:2 mix of lysis buffer and capturing hybridization buffer.
    11. Add magnetic Streptavidin beads to lysate and keep rotating for 1 h at 37 °C.
    12. Collect the beads using a magnetic rack.
    13. Wash beads three times for 5 min at 37 °C with low salt wash buffer (see Recipes).
    14. Wash beads two times for 5 min at 37 °C with high salt wash buffer (see Recipes).
    15. Wash beads three times for 5 min at 37 °C with low salt wash buffer.
    16. Elute the RNA from the beads by adding elution buffer (see Recipes) and boil at 95 °C for 5 min. Use approx. 0.75 µl TE buffer per µl Streptavidin bead slurry used in Step H10.
    17. Extract the RNA using an RNA extraction kit and determine the amount of captured RNA of targeting and non-targeting probes by qRT-PCR or Northern blot.

Data analysis

  1. Determining specificity and sensitivity of smFISH
    1. The rationale behind using an array of singly labeled probes to detect target transcripts is the local increase of signal on the specific RNA molecule–due to specific hybridization of the entire array–relative to the background resulting from e.g., random aspecific binding of individual probe molecules (Femino et al., 1998; Raj et al., 2008). Consequently, most of the aspecificity appears as background that can be filtered out (e.g., by the xsPT plugin during Step 7 of this section). In extreme cases, however, the probes may accumulate in specific subcellular structures/organelles, mainly in the nucleolus. If this is observed, we recommend increasing the stringency of the hybridization and the subsequent two wash steps (Steps 10-15 of Procedure F) by increasing the temperature and/or decreasing the salt concentration in the corresponding buffers. Decreasing the probe concentration and increasing the relative amount of the organic solvent (e.g., ethylene carbonate) could be also tried out, in this order (see Notes 16 and 18).
    2. To test whether the given smFISH setup–including the probe set and the applied protocol–is detecting all of the target molecules and only the target molecules, a two color smFISH reaction against the transcript of interest can be performed (Figures 3B-3B” and Video 1).

      Video 1. A short tutorial (~20 min) on how to use ImageJ and R to establish single molecule sensitivity of an smFISH experiment. A further tutorial on using the xsPT plugi-in is available in Gaspar et al., 2017.

    3. Split the unlabeled ssDNA oligonucleotides into two non-overlapping arrays and label them with two spectrally distinguishable fluorophores–e.g., Atto532 and Atto633–as described in Procedures B and C. (see Note 6)
    4. Carry out an smFISH experiment–e.g., as described in Procedure F–using the two sets of probes simultaneously.
    5. Acquire images of both fluorophores while avoiding cross-talk of the two channels–e.g., by performing sequential scan during confocal scanning microscopy.
    6. Optional: perform deconvolution on the resulting images to enhance signal to noise ratio and facilitate downstream analysis.
    7. Analyze the overlap of the two signals e.g., by using the xsPT plugin of ImageJ (Gaspar and Ephrussi, 2017).
    8. xsPT segments one of the channels (for more details, see Gaspar and Ephrussi, 2017) and performs tracking through an image sequence, e.g., a Z-stack allowing to recognize objects in 3D.
    9. Set the parameters of the plugin to detect all–or at least most of–the objects in the reference channel (for details, please refer Video S4 and S5 of Gaspar and Ephrussi, 2017). In the tracking part, set a maximum displacement of 1 px (the center of an object is not expected to shift much between the slices), set ‘Prefer short steps’ and uncheck ‘Manual tracking’ (i.e., perform automated tracking). Let the plugin run.
    10. RNPs–and other structures smaller than the diffraction limit–are expected to be observed in at least three consecutive slices when the image is four-fold over-sampled according to the modified Nyquist criterion (z-step size is typically between 180-300 nm). Remove trajectories that are shorter than three frames (‘Min. trajectory = 3’ and press ‘Filter trajectories’). You may further filter the recognized objects by selecting a region of interest in the image and pressing ‘Filter trajectories’.
    11. Save the filtered trajectories. The integrated signal intensities of both the reference and any other channels will be stored in the resulting .csv file. Four of such .csv files (Supplementary Files 4-7) are provided as examples. These–and any user generated files–can be analyzed using the smFISH_analysis.R script (Supplementary File 3).
    12. Determine a detection threshold for the reference channel (e.g., the minimum or the lowest 0.1th percentile of the integrated signal intensity).
    13. Repeat Steps A9-A12 using the other channel as the reference. This analysis results in independent measurements of the detection threshold for both channels.
    14. Test what fraction of the objects of the non-reference channel has higher signal intensity than the corresponding detection threshold, which was determined in the parallel analysis. If this fraction is over 80% for both channels, the smFISH analysis is considered to be at single molecule sensitivity (low false negative detection) and highly specific (low false positive detection rate) (Figure 3D).
    15. Lower than 80% co-detection of the same transcripts with the two probe sets–in extreme cases no overlap of the two signals or no signal at all–indicate insufficient sensitivity that is usually due to too high stringency during the hybridization and the two subsequent washes (Steps 10-15 of Procedure F). To overcome this problem, we recommend decreasing the temperature, increasing the salt concentration in the corresponding buffers as well as the probe concentration and decreasing the relative amount of the organic solvent during hybridization (try implementing these changes sequentially, in the listed order) (see Notes 16 and 18).
    16. Plotting the signal intensities of the reference and the non-reference channel can give insight into the RNA copy number of the objects (Figure 3C). In case of single RNA molecules, the two signals are not expected to correlate well as they are primarily governed by stochastic events (e.g., the hybridization efficiency of the probes, Figure 3D). If there are more than one copy of target transcripts in the objects, these stochastic events tend to average out yielding an improved linear relationship between the signals of the two channels which get stronger and stronger with the increase of the signal intensity.

  2. Determining the efficiency and specificity of RNA capture
    1. After RNA extraction, set up reverse transcription of the 0.1% input sample and eluted sample from the non-targeting and specific capture (see Note 15).
    2. From the cDNA, make a dilution series of the ‘input’ (e.g., 0.1%, 0.01%, 0.001% and 0.0001%) and dilute ‘captured’ sample to at least ten-fold. Set up triplicates of qRT-PCR reactions to amplify the target transcript and other, abundant non-target RNAs (e.g., the 18S ribosomal RNA) from the ‘input’ dilutions and the eluted ‘capture’ samples using appropriate PCR primer pairs.
    3. Calculate the amount of ‘captured’ RNA by comparing it to the regression of the diluted ‘input’ sample. For more details, see Gaspar et al., 2017b.

Notes

  1. You may use an argon-filled chamber. Another, simple means to obtain moisture free environment is to fill 50-100 g of silica gel into an about 20 x 20 x 20 cm polystyrene or plastic box with a lid.
  2. The smFISHprobe_finder.R script designs target specific antisense ssDNA oligonucleotides such that the terminal uracil nucleotide added during the labeling will become part of the hybrid. Any large gaps (> the double of minLength) between such probes are further scanned for potential probes that do not satisfy this terminal U rule but satisfy the other search criteria. Alternatively, the probe finder script can be used without the terminal U rule (lines 93-96 of smFISHprobe_finder.R script). Of note, we have found no adversary effect of a non-complimentary uracil nucleotide during hybridization at the 3’ end of the produced probes (data not shown).
  3. We found no practical differences between Amino-11-ddUTP or 5-propargylamino-ddUTP regarding dye conjugation and enzymatic incorporation of the resulting terminator nucleotide (Figure 2).
  4. We found that BDP-FL-NHS ester is quite hydrophobic and requires higher organic solvent concentration. When using this dye, add 1.8x DMSO before slowly titrating in the dye in Step A4 and skip Step A5. If precipitation occurs add more DMSO and calculate the final concentration accordingly.
  5. Originally, we added Tris-HCl, pH 7.5 to a 10 mM final concentration to quench the remaining NHS ester activity. However, we found no adverse effects of omitting this quenching step on the downstream enzymatic reaction.
  6. Although all oligonucleotides targeting the same transcript could be combined in one master-mix, we recommend preparing at least two non-overlapping mixtures, e.g., by combining every other oligonucleotides, by splitting them into 5’ and 3’ halves or grouping them by oligonucleotide length. Label these mixtures separately–this will facilitate troubleshooting, i.e., when the low degree of labeling is observed (Procedure E) and also allows benchmarking the smFISH performance (Procedure G). We typically order the oligos resuspended in H2O to 250 μM concentration.
  7. To facilitate drying, make sure that all residual ethanol is removed from the walls of the tube by a quick pulse of centrifugation after removing the bulk of the supernatant.
  8. To determine the degree of biotinylation, Colorimetric Biotin Assay Kit based on color loss of 2-(4-hydroxyphenylazo)-benzoic acid could be used. However, we found that incorporation of biotin-ddUTP is always 100% (see Figure 1 of Gaspar et al., 2017b) and therefore we do not routinely determine the degree of biotinylation.
  9. The organic dyes usually have also some absorption at 260 nm. The correction factor at 260 nm (cf260 nm) provided by the dye manufacturer specifies this absorbance as the fraction of the maximal absorption of the dye. This cf260 nm and the absorption of the dye at maximum allows for the correction of the OD260 nm value when calculating the oligonucleotide concentration.
  10. Steps 5-8 of Procedure F (smFISH) will result in a limited proteolysis and denaturation of proteins and RNA secondary structures and thus they may facilitate target recognition of the probes. However, if the fluorescence of fluorescent proteins (e.g., Emerald-GFP, as in Gaspar et al., 2017a) is to be preserved these steps should be omitted. In case of oskar mRNA, we found a minuscule effect of omitting these steps on the hybridization results.
  11. The final probe concentration we use is between 1-2.5 nM/probe. We find that lower concentrations may impair the sensitivity of the reaction while higher concentrations–although rarely they may cause staining artifacts–do not affect the quality of the hybridization but result in unnecessary waste of material.
  12. We found that 80% TDE boosts the brightness of GFP and red fluorescent proteins without affecting the signal of Atto565 and Atto633. However, the green and yellow fluorescent dye are substantially dimmer in this mounting medium and thus we recommend using e.g., VectaShield.
  13. If high amounts of starting material are required due to low abundance of target RNA, we use a method for mass-isolation of ovaries (similar to Jambor et al., 2015).
  14. The probe concentration should be adjusted depending on the amount of RNA present in your sample. We estimated the amount of the desired target RNA by qRT-PCR or Northern blot together with known standards of target RNA.
  15. Make sure not to oversaturate the RT reaction. The maximum volume is determined by the concentration of the ‘input’ sample.
  16. We recommend changing only one parameter at a time. It is a good practice to include the original value and two-three modified values of the parameter in these optimization steps. We find that once the listed parameters are optimized for the specimen, they are an excellent starting point for all smFISH reactions performed with yet uncharacterized probe sets, and in 95% of the cases those parameters provide good quality results.
  17. However, ‘aspecificity’ that arises from (partial) complementarity of a large fraction of the probe array to another transcript(s) may result in signal indistinguishable from that produced by the specific hybrids. This phenomenon can be only tested in control samples that lack the RNA-to-be-detected (RNA null mutants or efficient RNAi knock-downs). Since these control samples are not readily available in most cases, we recommend BLAST-ing the probe sequences against the host transcriptome during the design phase (before starting Procedure B) to rule out such cryptic targets.
  18. If an already tested and proven-to-work probe set results in such low sensitivity hybridization, the cause is likely nuclease contamination in one of the buffers. In such a case, it is recommended to replace all buffers used for the hybridization.

Recipes

  1. TE buffer
    10 mM Tris base
    1 mM EDTA
    Adjust pH to 8.0
  2. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Adjust pH to 7.4
    Store at RT
  3. Fixative
    10 ml 16% (v/v) EM-grade paraformaldehyde
    30 ml 1x PBS
    Sterile filter with 0.22 μm filter
    Store at 4 °C
  4. PBT
    0.1% Triton X-100 in 1x PBS
    Store at RT
  5. 1.5x PAGE loading buffer
    ¾ 8 M urea in 1x PBS
    ¼ 6x gel loading dye
    Store at RT
  6. 10x TBE
    1 M Tris base
    1 M boric acid
    20 mM EDTA
    Store at RT
  7. 15% PA - 8 M urea stock (200 ml)
    75 ml 40% (v/v) Acrylamide/Bis solution
    20 ml 10x TBE
    96 g urea
    Top up to 200 ml with dH2O
    Stir and heat (max 100 °C) until the urea completely dissolves
    Store at RT isolated from light
  8. 20x SSC
    3 M NaCl
    300 mM Na-citrate
    pH 7.0
    Store at RT
  9. 2x wash-HYBEC
    2x SSC
    15% (v/v) ethylene carbonate
    1 mM EDTA
    0.1% Triton X-100
    Store at RT
  10. 2x full-HYBEC
    2x SSC
    15% (v/v) ethylene carbonate
    1 mM EDTA
    50 µg/ml heparin
    100 µg/ml salmon sperm ssDNA
    0.1% Triton X-100
    Store at RT
  11. 80% TDE
    80% (v/v) 2,2’-thiodiethanol (Sigma-Aldrich)
    20% (v/v) 1x PBS
    Mix well and store at RT
  12. Lysis buffer
    50 mM Tris-HCl pH 7.0
    10 mM EDTA
    1% (v/v) SDS
    Fresh 1 mM PMSF
    Fresh cOmplete® mini EDTA-free protease inhibitor*
    1:2,000 fresh RiboLock RNAse Inhibitor
  13. Capturing hybridization buffer
    50 mM Tris-HCl pH 7.0
    750 mM NaCl
    1 mM EDTA
    1% (v/v) SDS
    15% (v/v) ethylene carbonate
    Fresh 1 mM PMSF
    Fresh cOmplete® mini EDTA-free protease inhibitor*
    1:2,000 fresh RiboLock RNAse Inhibitor
  14. Low salt wash buffer
    2x SSC
    0.5% (v/v) SDS
    Fresh 1 mM PMSF
    Fresh cOmplete® mini EDTA-free protease inhibitor*
  15. High salt wash buffer
    750 mM NaCl
    30 mM sodium citrate pH 7.0
    0.5% (v/v) SDS
    Fresh 1 mM PMSF
    Fresh cOmplete® mini EDTA-free protease inhibitor*
  16. Elution buffer
    10 mM Tris-HCl pH 7.0
    1 mM EDTA
    Store at RT

*Note: Add according the manufacturer’s instructions.

Acknowledgments

This work was funded by the EMBL. This protocol is adapted from Gaspar et al., 2017b. The authors declare no conflicts of competing interests.

References

  1. 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.
  2. Gaspar, I. and Ephrussi, A. (2015). Strength in numbers: quantitative single-molecule RNA detection assays. Wiley Interdiscip Rev Dev Biol 4(2): 135-150.
  3. Gaspar, I. and Ephrussi, A. (2017). Ex vivo ooplasmic extract from developing Drosophila oocytes for quantitative TIRF microscopy analysis. Bio-protocol 7(13).
  4. Gaspar, I., Sysoev, V., Komissarov, A. and Ephrussi, A. (2017a). An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs. EMBO J 36(3): 319-333.
  5. Gaspar, I., Wippich, F. and Ephrussi, A. (2017b). Enzymatic production of single-molecule FISH and RNA capture probes. RNA 23(10): 1582-1591.
  6. Jambor, H., Surendranath, V., Kalinka, A. T., Mejstrik, P., Saalfeld, S. and Tomancak, P. (2015). Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. eLife 4: e05003
  7. Khong, A., Matheny, T., Jain, S., Mitchell, S. F., Wheeler, J. R. and Parker, R. (2017). The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol Cell 68(4): 808-820 e805.
  8. Little, S. C., Sinsimer, K. S., Lee, J. J., Wieschaus, E. F. and Gavis, E. R. (2015). Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat Cell Biol 17(5): 558-568.
  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.
  10. Sage, D., Donati, L., Soulez, F., Fortun, D., Schmit, G., Seitz, A., Guiet, R., Vonesch, C. and Unser, M. (2017). DeconvolutionLab2: An open-source software for deconvolution microscopy methods-image processing for biologists. Methods 115: 28-41.

简介

短的,单标记的ssDNA寡核苷酸阵列使得能够与单分子灵敏度和有效的转录物特异性RNA捕获进行原位杂交。 在这里,我们描述了一个简单的酶促协议,可以使用基本的实验室设备将PCR寡核苷酸阵列以定量,成本高效和灵活的方式转换为smFISH和RAP探针组。

【背景】合成来源的多个单标记的短寡核苷酸的使用极大地改进了对特异性转录物的高特异性和单分子灵敏度的检测(Femino等人,1998; Raj等人。,2008)。这种探针分子与经典使用的长核酸探针相比具有改进的穿透性并且需要更温和的杂交条件,从而更好地保存标本的结构(例如,Little等人 >,2015,Gaspar 等,2017a)。由于在该设计中多个寡核苷酸 - 通常24-96-靶向相同转录物的不同部分,因此在非特异性背景上在特异性靶分子上发生信号累积,这与由长的多标记探针产生的相等信号相反(Raj ,2008)。此外,由于单个短探针的标记是定量的 - 与长探针的随机标记相反 - 信号强度与给定位点处的转录本拷贝数直接和线性相关,允许精确记录/计数靶RNA分子(Raj等人,2008,Little等人,2015)。到目前为止,smFISH探针阵列的生产依赖于化学合成和标记,使得这种单分子FISH应用不灵活且成本高昂。在这里,我们描述了利用末端脱氧核苷酸转移酶(TdT)和定制标记的终止子核苷酸将任何定制组装阵列转化成有效且成本有效的酶三锅式探针生产(3P <3>)测定法的廉价PCR寡核苷酸导入带有实验者选择的荧光或非荧光标记的smFISH探针(Gaspar等人,2017b)。这些酶促产生的3P3探针与其他来源的smFISH探针在化学上几乎相同。因此,可以使用相同的方案(针对正在研究的给定样本进行优化)来执行单分子FISH(Gaspar和Ephrussi,2015年综述)和RNA捕获分析(参见例如,Gaspar等人2017年和Khong 等人,2017年)。

关键字:末端脱氧核苷酸转移酶, 标记的终止子核苷酸, 探针生成, 单分子FISH(smFISH), RNA捕获, RNA亲和纯化(RAP)

材料和试剂

  1. 1.5ml Eppendorf管(例如,Sigma-Aldrich,目录号:Z336769)。
  2. 0.2ml薄壁PCR管(例如,Corning,目录号:6571)
  3. 2厘米厚的胶带(Tesa)
  4. 玻璃载玻片(例如,VWR,目录号:631-0411)和盖玻片( eg ,22×22×0.17mm,Marienfeld-Superior,目录号:0107052)样品制备

  5. 15ml试管(例如,Corning,Falcon ,产品目录号:352097)
  6. 0.22微米的过滤器(例如,Corning,目录号:431227)
  7. 3厘米宽的折叠式回形针( ,Staples,产品目录号:WW-9130156)
  8. 胺反应性标签(测试和工作):
    1. BDP-FL-NHS(Lumiprobe,目录号:11420)
    2. Atto-tec Atto488-NHS(Atto-tec,目录号:AD488-31),Atto532-NHS(Atto-tec,目录号:AD532-31),Atto565-NHS(Atto-tec,目录号:AD565 -31)和Atto633-NHS(Atto-tec,目录号:AD 633-31)
    3. AlexaFluor488-NHS(Thermo Fisher Scientific,Invitrogen TM,目录号:A20000)
    4. Abberior STAR 470SXP-NHS(Abberior,目录号:1-0101-008-3)和Abberior STAR RED-NHS(Abberior,目录号:1-0101-011-3)
    5. 生物素-NHS(Sigma-Aldrich,目录号:H1759)
  9. 无水DMSO(例如,Sigma-Aldrich,目录号:276855)
  10. 可选:硅胶(如,Merck,产品目录号:1.01969.1000)(见注1)
  11. 氨基-11-ddUTP(Lumiprobe,目录号:15040)或5-炔丙基氨基-ddUTP(Jena Biosciences,目录号:NU-1619)
  12. 1M NaHCO 3 3,pH 8.4(例如,Sigma-Aldrich,目录号:S5761)。
  13. 定制设计的非特异性ssDNA寡核苷酸目标特定阵列(脱盐纯化就足够了,参见设计软件部分)
  14. 20U /μl含有5xTdT缓冲液(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0161)的末端脱氧核苷酸转移酶(TdT)
  15. 1-3M乙酸钠,pH5.5(例如,Sigma-Aldrich,目录号:S2889)。
  16. 5mg / ml直链丙烯酰胺(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:AM9520)。
  17. 乙醇(例如,Merck,EMD Millipore,目录号:1.00983)
    1. 100%乙醇,-20°C
    2. 80%乙醇,4℃
    3. 70%乙醇,RT
  18. 不含核酸酶的ddH 2 O(例如,New England Biolabs,目录号:B1500S)
  19. 40%丙烯酰胺/双溶液,29:1(例如Bio-Rad Laboratories,目录号:1610146)。
  20. 尿素(例如,,Sigma-Aldrich,目录号:U5378)
  21. N,N,N',N' - 四甲基乙二胺(TEMED)(例如,Sigma-Aldrich,目录号:T9281)。
  22. 10%(w / v)过硫酸铵(APS)(例如,Sigma-Aldrich,目录号:A3678)。
  23. 6x凝胶加样染料(例如,新英格兰生物实验室,目录号:B7021S)。
  24. SYBR-GOLD(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:S11494)。
  25. 可选:比色生物素测定试剂盒(比如,Sigma-Aldrich,目录号:MAK171)(见注8)
  26. 20mg / ml蛋白酶-K(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:AM2546)。
  27. 安装介质
    1. VectaShield(Vector Laboratories,目录号:H-1000)
    2. 80%TDE(见食谱)
  28. Pierce Amidin琼脂糖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:20219)
  29. Dynabeads MyOne TM C1(Thermo Fisher Scientific,Invitrogen TM,目录号:65001)
  30. Quick-RNA TM MicroPrep试剂盒(Zymo Research,目录号:R1050)
  31. Tris-HCl pH 7.0(例如,Sigma-Aldrich,Roche Diagnostics,目录号:10812846001)。
  32. 比色生物素测定试剂盒(Sigma-Aldrich,目录号:MAK171)
  33. Tris碱(例如,Sigma-Aldrich,目录号:T1503)
  34. 乙二胺四乙酸(EDTA)(例如,Sigma-Aldrich,目录号:E5391)
  35. 氯化钠(NaCl)(如emck,Merck,目录号:106404)
  36. 氯化钾(KCl)(如emck,Merck,目录号:104936)
  37. 磷酸二氢钾二水合物(KH2PO4•2H2O)(例如Merck,产品目录号:
  38. 磷酸二氢钠(Na 2 HPO 4)(例如Merck,产品目录号:106342)
  39. EM级多聚甲醛(例如,Electron Microscopy Sciences,目录号:15710)
  40. Triton X-100(例如,Sigma-Aldrich,目录号:X100)
  41. 硼酸(如emck,Merck,目录号:100165)
  42. 碳酸亚乙酯(例如,Sigma-Aldrich,目录号:E26258)
  43. 50mg / ml肝素(例如,Sigma-Aldrich,目录号:H3393)。
  44. 10mg / ml鲑鱼精子DNA(例如,Sigma-Aldrich,目录号:D7656)。
  45. 2,2'-硫代二乙醇(Sigma-Aldrich,目录号:166782)
  46. 20%(v / v)SDS(例如,Sigma-Aldrich,目录号:05030)。
  47. PMSF(如,Sigma-Aldrich,目录号:P7626)
  48. cOmplete mini mini EDTA无蛋白酶抑制剂(Roche Diagnostics,产品目录号:11836170001)
  49. RiboLock RNA酶抑制剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0381)
  50. 柠檬酸钠(例如,Sigma-Aldrich,目录号:S1804)
  51. TE缓冲液(见食谱)
  52. 1x PBS(见食谱)
  53. 固定剂(见食谱)
  54. PBT(见食谱)
  55. 1.5倍的PAGE加载缓冲区(见食谱)
  56. 1x和10x TBE(见食谱)
  57. 15%PA - 8 M尿素(见食谱)
  58. 20x SSC缓冲液(见食谱)
  59. 2次全HYBEC(见食谱)
  60. 2次洗涤 - HYBEC(见食谱)
  61. 裂解缓冲液(见食谱)
  62. 捕获杂交缓冲液(见食谱)
  63. 低盐洗涤缓冲液(见食谱)
  64. 高盐洗涤缓冲液(见食谱)
  65. 洗脱缓冲液(见食谱)

设备

  1. 可选:惰性气体(例如,Argon)手套箱(例如,惰性技术,型号:PureLab HE 2GB)(见注1)
  2. 带有可编程热盖的PCR仪(如,Bio-Rad Laboratories,目录号:1851148)
  3. -20°C冷冻机
  4. 冷藏台式离心机(例如,Eppendorf,目录号:5426000018)
  5. 锥形瓶
  6. Handcast PAGE系统,包括1mm间隔板(例如,Bio-Rad Laboratories,产品目录号:1653311),短板(例如Bio-Rad Laboratories,目录号:1653308)和15孔梳(例如Bio-Rad Laboratories,目录号4560016)。
  7. 垂直电泳细胞(例如,Bio-Rad Laboratories,目录号:1658005)
  8. 电泳电源(例如,Bio-Rad Laboratories,目录号:1645050)
  9. 凝胶文件系统,带有过滤器,用于对SYBR-GOLD的荧光和用于标记的荧光染料(例如,Bio-Rad Laboratories,目录号:17001402)进行成像。
  10. P2,P200和P1000移液器
  11. 摇摆式热电偶( ,Eppendorf,型号:ThermoMixer®C,产品目录号:5382000015)
  12. 用于成像的显微镜(我们使用装配有63x NA = 1.4油浸物镜和两个HyD检测器的Leica SP8(Leica,型号:Leica TCS SP8))
  13. 组织研磨机(例如,DWK生命科学公司,Kimble,产品目录号:8853000015或8853000040)
  14. 旋转器(,例如,Cole-Parmer,Stuart,型号:Rotator SB3)
  15. 磁力架(例如,新英格兰生物实验室,目录号:S1507S)
  16. 无潮湿室(见注1)
  17. UV / VIS分光光度计(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 8000,目录号:ND-8000-GL )
  18. Nutator(,例如,Labnet International,型号:S0500)

软件

  1. 探针设计器算法,例如,Stellaris TM探针设计器
    https://www.biosearchtech.com/support/tools/设计软件/ stellaris-probe-designer ,需要注册)或提供的 smFISHprobe_finder.R 脚本(补充文件1 ,请参阅注释2和17)
  2. MS Office Excel运行交互式 probe_calculator.xls 工作表(补充文件2
  3. ImageJ / FIJI( https://imagej.nih.gov/ij/ )与xsPT插件( https://github.com/Xaft/xs/blob/master/_xs.jar
  4. 可选:解卷积软件,例如,Huygens Essentials( https://svi.nl/Huygens- Essential )或DeconvolutionLab2(Sage et al。,2017; http ://bigwww.epfl.ch/deconvolution/deconvolutionlab2/
  5. R(优先与RStudio)进行数据分析
  6. smFISH_analysis.R 来分析smFISH的敏感性和特异性(补充文件3

程序



图1.三锅式探针生产测定的图形概述。 :一种。标签与NH2-ddUTP终止子核苷酸偶联; B.TdT介导的ssDNA寡核苷酸的标记; C.纯化标记的ssDNA分子(探针)。

  1. 制备染料结合的ddUTP(第一个罐,图1A)
    1. 在无水环境中将染料-NHS酯在无水DMSO中重建至40mM最终浓度。 (见注1)
    2. 在干净的1.5ml Eppendorf管中,等分Xμl20mM氨基-11-ddUTP或5-炔丙基氨基-ddUTP。 (见注3)
    3. 加入0.2Xμl的1M NaHCO 3(pH = 8.4)。
    4. 最后,加入40μl染料-NHS酯(见注4)。充分混合并在室温孵育2小时,避光。
    5. 孵育后,加入1.8XμldH 2 O 0将反应体积扩大至4Xμl。这导致5mM库存的染料结合ddUTP(参见注释5)。
      所产生的标记核苷酸在-20°C储存一年内稳定。

  2. 生产标记的ssDNA寡核苷酸(第二个罐,图1B)
    1. 准备应标记在一起的所有不同ssDNA寡核苷酸的等摩尔混合物,即靶向相同转录物的 。寡核苷酸的总浓度不应低于100μM。 (见注6)
    2. 要标记1 nmol的寡核苷酸混合物,请在0.2 ml薄壁PCR管中制备以下标记混合物:
      请使用交互式的 probe_calculator.xls Excel工作表(补充文件2 ),将反应组合物调整为用于标记的染料-ddUTP。

    3. 在热盖设置为37°C的PCR仪中,在37°C O / N(16-18 h)下孵育。

  3. 标记的寡核苷酸混合物的纯化和光谱分析(第三个罐,图1C)
    1. O / N孵育后,向反应混合物中加入60μl1M醋酸钠(pH = 5.5),125μldH 2 O和1.5μg直链丙烯酰胺。
    2. 将整个200μl转移到干净的1.5ml Eppendorf管中。加入800μL预冷的-20°C的100%乙醇。
      翻转管几次,并将其放入-20°C冰箱约15-20分钟。
    3. 同时,将台式离心机冷却至4°C。
    4. 在-20℃温育后,在4℃下将寡核苷酸混合物在16,000×gg下旋转20分钟。
    5. 取出上清液并加入1ml预冷的4%的80%乙醇。漩涡直到颗粒从管底部分离。
    6. 在4℃下以16,000×g g离心5分钟。
    7. 用1 ml 80%乙醇将试管中的沉淀洗去,并将包括浮动沉淀在内的整个体积转移到干净的1.5 ml Eppendorf试管中。重复步骤C5和C6两次。
    8. 从最后一次清洗中取出上清液,让沉淀物在空气中干燥。 (见注7)

    9. 在15-50μl不含核酸酶的dH sub 2 O中重悬干燥的颗粒。
    10. 测量标记的寡核苷酸混合物在260nm和最大染料吸收(例如,对于Atto565,570nm)的吸光度。
    11. 为了计算浓度和标记程度(DOL),在260nm下测量未标记的,未稀释的寡核苷酸混合物的吸光度。 (见注8)
    12. 通过将测得的OD 260nm值与混合物浓度(以M计)相除来计算寡核苷酸混合物的摩尔消光系数(εoligo)。将此值增加9,000 mol -1 -1 sup -1以校正加入到ssDNA分子3'中的UTP。
    13. 通过将染料校正的OD 260nm吸收除以ε寡核苷酸来计算标记的寡核苷酸的浓度:c =寡核苷酸=(OD n) (参见注释9)。
      260nm-cf 260nm×OD染料)/ε寡核苷酸(见注9)。
    14. 计算染料的浓度如下:染料= OD染料/染料染料(由染料生产商提供)。
    15. 通常,只有一小部分染料分子作为污染物存在(即,不含ssDNA),因此DOL估计如下:DOL = c染料/ c oligo 。
    16. 回收的寡核苷酸的比例通过将回收的寡核苷酸与初始量的比例(回收率=初始寡核苷酸×重新悬浮液/初始重量% / sub>)。
    17. 测量的OD值可以输入到交互式的 probe_calculator.xls Excel工作表中( Supplementary File 2 )以获得标记的寡核苷酸混合物的浓度和DOL。
    18. 将标记的探针储存在-20°C。

  4. 3P <3>探针生产的故障排除
    标记的具有0.9 < DOL≤1.0被认为是可用于smFISH应用的优质产品。
    1. DOL低于0.9的最典型原因是寡核苷酸混合物的组成。我们目前还不了解混合物的哪些性质(例如,混合物中分子内和分子间杂化体的形成)会影响标记效率。但是,如果DOL&lt; 0.9时,我们推荐将寡核苷酸混合物“分裂”成2-3个非重叠部分,即制备原始混合物中存在的ssDNA寡核苷酸的2-3个混合物。这些混合物的单独标记可以帮助识别在标记反应中表现异常的任何分子。在大多数情况下,我们发现这种“分裂”几乎完全缓解了低标记效率问题,即每种分裂混合物的DOL增加到0.9以上。
    2. 我们发现的另一个解决方案是重新标记已经标记的寡核苷酸混合物,即开始通过使用标记的ssDNA分子作为输入的来自步骤B2的标记方案。 (REF)
    3. 低DOL的第二个最常见原因是未标记的寡聚体混合物的摩尔消光系数的测量结果不准确(低于实际值)。这可以注意到,当计算回收的寡核苷酸的比例(回收率%)时,获得高于1的值。在这种情况下,我们建议重新测量不同浓度( eg ,50,100和200μM)的未标记寡核苷酸混合物的OD 260nm,并估计ε通过取适合数据点的线的斜率来确定 oligo 。
    4. 我们发现TdT的末端转移酶活性因批次而异。当使用1倍标准量的酶时,这可能导致不适当的标记(DOL <0.9)。我们建议在开始一个新的TdT等分试样时重新定义标准量,方法是使用递增浓度的TdT 例如设置单个ssDNA寡核苷酸与Atto633-ddUTP(程序B和C) ,配方生成器中标准量的1倍,2倍和3倍。使用产生DOL>的酶的最小浓度> 0.9作为新的标准金额。
    5. DOL&gt; 1.0也可能指示不精确确定的ε寡核苷酸。这将表现为寡核苷酸的巨大损失(恢复<50%)。尝试与步骤D3中相同的解决方案。
    6. 如果ε oligo 是合适的,则DOL> 1.0表示自由染料污染。在这种情况下,建议通过PAGE测量DOL(程序E)。如果通过密度测定法测定的DOL是〜1.0并且两种DOL的差异很小(最大0.05-0.1),则在下游应用中游离染料污染被认为是无害的。如果它更高,推荐通过另一种方法(例如,通过尺寸排阻色谱法)重新纯化寡核苷酸混合物。当使用Abberior470SX-,Atto488-和AlexaFluor488-ddUTP时(Gaspar等人,2017b),我们观察到如此高的游离染料污染。

  5. 标记的寡核苷酸的PAGE分析
    凝胶电泳提供了一种简单的方法来确认从光谱数据估计的DOL,并在非荧光修饰(例如,生物素化)的情况下计算DOL。 PAGE分析还允许对大多数标记的ddUTP类似物质量进行质量控制,以确定染料结合的ddUTP原液中是否存在未偶联的ddUTP分子(在程序A中产生),这会导致DOL降低的寡核苷酸。
    重要:只应通过PAGE分析含有相同长度(例如仅20个)的ssDNA分子的寡聚物混合物。
    1. 用70%乙醇擦拭凝胶铸件清洁玻璃板,组装盒子并密封20毫米宽的透明胶带,打开顶部(梳子将插入的位置)。将两个折叠式夹子放在盒子的两侧,使夹子夹在两块玻璃板之间的间隔物上方。
    2. 要浇铸10×8厘米的丙烯酰胺凝胶,添加8毫升15%PA - 8 M尿素储备(请参阅食谱),40微升10%APS和5微升TEMED到一个锥形瓶中。充分摇匀烧瓶并将混合物倒入组装好的盒子的两块板之间。填充到顶部。
    3. 插入梳子,水平放置盒子,等待凝胶聚合(15-20分钟)。
    4. 卸下夹子,胶带和梳子。用dH2O冲洗盒的外部以去除在外表面上聚合的凝胶片并组装PAGE室。
    5. 填充1x TBE缓冲液(见配方),用2.5 mA / cm电流(20 mA,8 cm长的凝胶)预凝胶30分钟。
    6. 同时,通过混合3μl1.5x PAGE上样缓冲液(参见食谱)和2μl含15-60 pmoles标记寡核苷酸的寡核苷酸混合物来制备5μl的每种样品。由于大小标记使用未标记的寡核苷酸混合物的稀释行(例如1.5cm 3和6pmol),所以不需要沸腾。
    7. 预运行(E5)后,使用P200移液器用1x TBE冲洗凝胶上的所有孔,以除去积聚的尿素,以防止加载。
    8. 装入样品并运行凝胶,直到二甲苯蓝(蓝色)和溴酚蓝(紫色)标记(来自6x凝胶上样染料)达到凝胶长度的三分之一和三分之二。
    9. 用凝胶成像仪对荧光标记的分子池进行成像,使用适当的滤光片组激发并检测掺入的荧光染料。

    10. 在1x TBE中用1:10,000稀释的SYBR-GOLD(或类似的RNA / ssDNA染料)孵育凝胶10-15分钟。
    11. 重新凝胶图像以检测未修饰和修饰的ssDNA库。
    12. 由于添加了大体积的终止子核苷酸,标记的寡核苷酸运行较慢,因此它们在PAGE期间与其未修饰的同位体充分分离。未修饰寡核苷酸的量可以通过比较相应的SYBR-GOLD荧光强度与用作上样对照的未标记寡核苷酸混合物稀释行的强度来测量(图2A和2A')。
    13. 未缀合的ddUTP的存在将导致产生未标记的,ddUTP终止的寡核苷酸。在凝胶上,它们表现为在未标记和标记的寡核苷酸荧光池之间迁移的中间条带。


      图2.标记的寡核苷酸混合物的PAGE分析。使用Atto633-氨基-11-ddUTP(泳道4)或Atto633-5-炔丙基氨基-ddUTP(泳道5)标记osk20nt-15x(Gaspar等人,2017b)探针混合物)。与未标记的寡核苷酸(绿色,A,灰色,A')相比,荧光标记的ssDNA物种(洋红色,A)在凝胶中迁移较慢。请注意,标有远红色荧光的探针分子在SYBR-GOLD通道中不可见,可能是因为从SYBR GOLD到Atto633的非常有效的能量转移形式可以淬灭SYBR GOLD荧光。当其他远红染料(例如,Abberior-RED)也是如此,当没有淬灭染料存在时(例如,生物素化探针)或当FRET荧光可以通过SYBR GOLD成像设置(例如,在Atto565的情况下)检测到,标记的探针出现在SYBR GOLD通道中(Gaspar et。,2017b )。将1.5,3,6,20和20pmol ssDNA混合物分别加载到1-5泳道。在与任一终止子核苷酸进行标记反应后,仅有少量(≤1.5pmol)未标记的寡核苷酸留在混合物中(泳道4和5A和5A'),表明用两种终止子核苷酸进行近定量标记。红色箭头表示溴酚蓝染料的荧光。

  6. 果蝇卵巢中的单分子FISH
    1. 将卵巢解剖成含有300-500μl固定剂的1.5ml Eppendorf管(卵巢解剖,请参阅Gaspar和Ephrussi,2017)。

    2. 通过章动解剖的材料修复20分钟
    3. 取出固定剂,用1毫升PBT冲洗卵巢(见食谱)。

    4. 在1毫升新鲜PBT中清洗卵巢10分钟
    5. 取代PBT并将蛋白酶-K加至2μg/ ml终浓度。在室温(RT)下固定5分钟。 (见注10)

    6. 在PBS中预热0.5 ml 0.05 v / v%SDS(见配方)至95°C
    7. 5分钟蛋白酶-K消化后,取出PBT并立即将预热的SDS / PBS施用于卵巢。在95°C孵育5分钟。

    8. 加入1 ml RT PBS至卵巢以冷却溶液。
    9. 用200μl2x全HYBEC取代洗液(见食谱)。
    10. 将试管转移至37-42℃的摇摆热块,并以1000RPM摇动10分钟。
    11. 同时,在50μl2x全HYBEC中制备探针溶液(5-12.5 nM /单个探针)。将探针溶液转移到加热块并让其预热(2-3分钟)。 (见注11)
    12. 在卵巢孵育10分钟后,通过将探针溶液混合到样品上已经存在的200μl2x全-HYBEC中来施用探针溶液。

    13. 在37-42°C孵育1.5-3小时,同时以1,000 RPM摇摆。
    14. 孵育结束前10-15分钟,预热两次1ml 2次洗涤-HYBEC(参见食谱)至杂交温度。
    15. 去除杂交溶液 - 它可以保持在4°C进行另一次杂交 - 并用1毫升预热的2x洗涤液 - HYBEC洗2×15分钟。

    16. 用1 ml RT PBT再洗涤一次15分钟。
    17. 彻底清除PBT并使用安装介质,例如,80%TDE(见食谱)或VectaShield。 (见注12)
    18. 在允许安装介质浸泡至少1小时后,将卵巢安装到载玻片上并使用高NA(> 1.1NA)目标(图3A)对其进行成像。


      图3.在发育中的果蝇卵室中oskar mRNA的smFISH分析A. oskar mRNA在转录活性护士细胞中产生(左)并将其运送到卵母细胞中,最终在后极处定位(在右侧)。女性生殖系细胞(护士细胞+卵母细胞)被一层体细胞上皮细胞包裹,卵泡细胞不表达oskar mRNA。 B.通过两种不同探针组osk37x-Atto532(绿色,osk3UTR#1-15 + oskCD#1-22)和oskCD20x-633检测到在A. oskar mRNA中的盒装区域的特写(红色,oskCD#23-42,加斯帕尔等人,2017b)。在护士细胞区室(B)中两个点样信号存在高度的共定位,并且在毛囊细胞(B-B“)中没有检测到这种强烈的探针积聚。箭头表示护士细胞的多倍体细胞核内的多个转录基因座(B)。比例尺表示10微米。 C和D.两个通道的信号强度的相关性。 C.当考虑蛋室中的所有物体时,建立强烈的线性相关性(R 2 = 0.972),表明检测到的物体的RNA含量是可变的。通过将多个高斯函数拟合到信号强度分布上(Little等人,2015; Gaspar等人,2017b),可以过滤含有单个拷贝的 oskar RNA。 D.在这种情况下,信号强度的相关性是低到中(R 2 = 0.264),然而绝大多数的目标可以在两个通道中被检测到 - 例如,在Atto633通道中分析的对象中,88.6%也可以在Atto532通道中检测到。垂直和水平黑色虚线分别代表Atto633和Atto532通道的检测阈值。红色虚线显示RNA(C)的单个和多个拷贝的物体之间的边界。颜色表示两个通道之间的相对强度差异(参见面板C中的键)。

      smFISH对象的数量(实验数量)用C和D表示
  7. 来自卵巢裂解物的RNA捕获
    1. 从PBS喂养2-3天龄的果蝇中分离卵巢(见注13)。
    2. 取出PBS并用3倍体积的裂解缓冲液重悬。
    3. 使用组织研磨机械均化。
    4. 转移到新鲜的15ml试管中并通过离心(140微克×克5分钟)清除裂解物。
    5. 将上清转移至新鲜试管中,用2倍体积的杂交缓冲液稀释(参见食谱)。
    6. 通过加入1:50 v / v Pierce 抗生物素蛋白琼脂糖进行预清洗,预先用1:2裂解缓冲液混合并捕获杂交缓冲液。
    7. 在室温下保持旋转器30分钟。
    8. 通过离心除去抗生物素蛋白琼脂糖(140微克×克5分钟)。
    9. 将预先裂解的裂解物(保留0.1%作为输入样品)分开,并用每毫升卵巢靶向和非靶向对照生物素化DNA探针0.25μg(参见注释14)补充并在旋转器上在37℃孵育2小时。
    10. 预洗3.75μl磁性链霉抗生物素蛋白珠粒/ ml溶胞产物,混合裂解缓冲液和捕获杂交缓冲液1:2。
    11. 将磁性链霉抗生物素蛋白珠子加入到裂解物中并在37℃下保持旋转1小时。

    12. 使用磁力架收集珠子

    13. 在37°C用低盐洗涤缓冲液洗珠三次,每次5分钟(见食谱)。
    14. 在37°C用高盐洗涤缓冲液洗两次珠子5分钟(见食谱)。

    15. 在37°C用低盐洗涤缓冲液洗三次珠子5分钟
    16. 通过加入洗脱缓冲液(参见食谱)从珠子中洗脱RNA,并在95℃煮沸5分钟。使用约。
      在步骤H10中使用0.75μlTE缓冲液/μl链霉亲和素珠浆液。
    17. 使用RNA提取试剂盒提取RNA,并通过qRT-PCR或Northern印迹确定捕获的靶向和非靶向探针的RNA量。

数据分析

  1. 确定smFISH的特异性和敏感性
    1. 使用一系列单标记探针检测靶转录物的原理是,由于整个阵列的特异性杂交,特定RNA分子上的信号相对于由例如产生的背景的局部增加,个体探针分子的随机非特异性结合(Femino等人,1998; Raj等人,2008)。因此,大多数非特定性显示为可以被滤除的背景(,例如,由本节的第7步中的xsPT插件)。然而,在极端情况下,探针可能积聚在特定的亚细胞结构/细胞器中,主要存在于核仁中。如果观察到这种情况,我们建议通过提高温度和/或降低相应缓冲液中的盐浓度来提高杂交的严谨性和随后的两个洗涤步骤(程序F的步骤10-15)。也可按此顺序尝试降低探针浓度并增加有机溶剂的相对量(例如,碳酸亚乙酯)(参见注释16和18)。
    2. 为了测试给定的smFISH装置(包括探针组和应用的方案)是否检测到所有的靶分子和仅靶分子,可以进行针对感兴趣的转录物的双色smFISH反应(图3B-3B“和视频1)。

      视频1

    3. 将未标记的ssDNA寡核苷酸分成两个不重叠的阵列,并按照程序B和C中所述将两个光谱可区分的荧光团标记为例如Atto532和Atto633(参见注释6)。
    4. 按照程序F所述进行smFISH实验 - 例如,同时使用两套探针。
    5. 通过在共聚焦扫描显微镜过程中执行顺序扫描,获取两种荧光团的图像,同时避免两个通道的串扰 - 例如。
    6. 可选:对结果图像执行解卷积以提高信噪比并促进下游分析。
    7. 通过使用ImageJ的xsPT插件(Gaspar和Ephrussi,2017),分析两个信号的重叠(例如)。
    8. xsPT将其中一个通道分段(有关更多详细信息,请参阅Gaspar和Ephrussi,2017),并通过图像序列执行跟踪,例如,一个允许识别3D对象的Z-stack。
    9. 设置插件的参数以检测参考通道中的全部或至少大部分物体(详情请参阅2017年Gaspar和Ephrussi的视频S4和S5)。在跟踪部分中,设置1px的最大位移(物体的中心在切片之间预计不会有太大偏移),设置'选择短步骤'并取消选中'手动跟踪'( ie ,执行自动跟踪)。让插件运行。
    10. 当图像根据改进的奈奎斯特准则进行四倍过采样时,RNP-以及小于衍射极限的其他结构 - 预计将在至少三个连续切片中被观察到(z步长通常在180-300nm之间)。删除短于三帧的轨迹('最小轨迹= 3'并按'过滤器轨迹')。您可以通过在图像中选择一个感兴趣的区域并按'过滤器轨迹'来进一步过滤识别的对象。
    11. 保存已过滤的轨迹。参考和任何其他通道的积分信号强度将存储在生成的.csv文件中。这样的.csv文件的四(补充文件4-7 )作为示例提供。可以使用 smFISH_analysis.R 脚本分析这些(以及任何用户生成的文件)(补充文件3 )。
    12. 确定参考通道的检测阈值(,例如,积分信号强度的最小或最低百分位数)。
    13. 使用另一个通道作为参考重复步骤A9-A12。该分析结果可以独立测量两个通道的检测阈值。
    14. 测试非参考通道的哪些部分物体具有比在并行分析中确定的相应检测阈值更高的信号强度。如果这两个通道的分数超过80%,smFISH分析被认为是单分子灵敏度(低假阴性检测)和高特异性(低假阳性检测率)(图3D)。
    15. 低于80%的两个探针组共同检测相同的转录物 - 在极端情况下,两个信号没有重叠或完全没有信号 - 表明灵敏度不足,这通常是由于杂交过程中过高的严格度以及随后的两个洗涤(程序F的步骤10-15)。为了克服这个问题,我们建议降低温度,增加相应缓冲液中的盐浓度以及探针浓度,并减少杂交过程中有机溶剂的相对量(尝试按照所列顺序依次实施这些变化)(见注16和18)。
    16. 绘制参考信号和非参考信道的信号强度可以深入了解对象的RNA拷贝数(图3C)。在单个RNA分子的情况下,这两种信号预计不会很好地相关,因为它们主要由随机事件(例如,探针的杂交效率,图3D)控制。如果对象中有多个目标转录本,那么这些随机事件倾向于平均,两个通道信号之间的线性关系会随着信号强度的增加而变得越来越强。

  2. 确定RNA捕获的效率和特异性
    1. RNA提取后,设置0.1%输入样品的逆转录,并从非靶向和特异性捕获中洗脱样品(参见注释15)。
    2. 从cDNA中,对'输入'(例如,0.1%,0.01%,0.001%和0.0001%)进行稀释系列并将'捕获'样品稀释至少10倍。设置qRT-PCR反应的一式三份以从“输入”稀释液和洗脱的“捕获”样品中扩增靶转录物和其他丰富的非靶RNA(例如,18S核糖体RNA)合适的PCR引物对。
    3. 通过将其与稀释的“输入”样品的回归进行比较来计算“捕获的”RNA的量。有关更多详情,请参阅Gaspar 等,2017b。

笔记

  1. 您可以使用充满氩气的腔室。另一种获得无水环境的简单方法是将50-100克硅胶填充到约20×20×20厘米的带有盖子的聚苯乙烯或塑料盒中。
  2. smFISHprobe_finder.R 脚本设计靶向特异性反义ssDNA寡核苷酸,使得在标记期间添加的末端尿嘧啶核苷酸将成为杂交体的一部分。对这些探针之间的任何大的间隙(&lt; minLength 的两倍)进一步扫描不满足该终端U规则但满足其他搜索标准的潜在探针。或者,可以使用探针查找程序脚本,而不使用终端U规则( smFISHprobe_finder.R 脚本的第93-96行)。值得注意的是,我们发现在产生的探针的3'末端杂交期间没有互补的尿嘧啶核苷酸的对抗效应(数据未显示)。
  3. 我们发现氨基-11-ddUTP或5-炔丙基氨基-ddUTP在染料结合和酶促掺入所得终止子核苷酸方面没有实际差异(图2)。
  4. 我们发现BDP-FL-NHS酯非常疏水,需要更高的有机溶剂浓度。当使用这种染料时,在步骤A4的染料中缓慢滴定之前加入1.8倍DMSO,并跳过步骤A5。如果发生降水,请添加更多的DMSO并相应计算最终浓度。
  5. 最初,我们加入Tris-HCl,pH 7.5至终浓度为10 mM以终止剩余的NHS酯活性。然而,我们发现省略这个淬火步骤对下游酶促反应没有不利影响。
  6. 尽管所有靶向相同转录产物的寡核苷酸都可以在一种主混合物中组合,但我们建议通过将其他寡核苷酸组合成至少两种非重叠混合物(例如),将它们分成5'和3将它们按寡核苷酸长度分成两半或两组。分开标记这些混合物 - 当观察到低程度的标记(程序E)并且还允许基准smFISH性能(程序G)时,这将便于故障排除,即。我们通常将重新悬浮于H 2 O中的寡核苷酸订购至250μM浓度。
  7. 为了促进干燥,在去除大部分上清液后,确保通过快速离心脉冲将所有残留的乙醇从管壁除去。
  8. 为了确定生物素化程度,可以使用基于2-(4-羟基苯偶氮) - 苯甲酸的颜色损失的比色生物素测定试剂盒。然而,我们发现生物素-ddUTP的掺入总是100%(参见Gaspar等人的图1,2017b),因此我们不常规地确定生物素化程度。
  9. 有机染料通常在260nm处也有一些吸收。由染料制造商提供的在260nm(cf260nm)的校正因子将该吸光度指定为染料的最大吸收的分数。当计算寡核苷酸浓度时,该最大值260nm和染料的吸收允许校正OD 260nm值。
  10. 程序F(smFISH)的步骤5-8将导致蛋白质和RNA二级结构的有限蛋白水解和变性,并且因此它们可以促进探针的目标识别。然而,如果要保存荧光蛋白(例如,Emerald-GFP,如Gaspar等人,2017a)的荧光,则应省略这些步骤。在 oskar mRNA的情况下,我们发现省略这些步骤对杂交结果的影响很小。
  11. 我们使用的最终探针浓度在1-2.5 nM /探针之间。我们发现较低的浓度可能会损害反应的敏感性,而较高的浓度(尽管很少会导致染色伪像)不会影响杂交的质量,但会导致不必要的材料浪费。
  12. 我们发现80%的TDE增加了GFP和红色荧光蛋白的亮度,而不影响Atto565和Atto633的信号。然而,绿色和黄色荧光染料在该安装介质中基本上较暗,因此我们推荐使用,例如,VectaShield。
  13. 如果由于靶RNA的丰度很低而需要大量的起始材料,我们使用一种大规模分离卵巢的方法(类似于2015年的Jambor等人, )。
  14. 探针浓度应根据样本中存在的RNA量进行调整。我们通过qRT-PCR或Northern印迹以及已知的靶RNA标准来估计所需靶RNA的量。
  15. 确保不要使RT反应过饱和。最大体积由“输入”样本的浓度决定。
  16. 我们建议一次只更改一个参数。在这些优化步骤中包含原始值和参数的两三个修改值是一种很好的做法。我们发现,一旦列出的参数针对样本进行了优化,对于使用尚未表征的探针组进行的所有smFISH反应,它们都是一个很好的起点,并且在95%的情况下,这些参数提供了优质的结果。
  17. 然而,由大部分探针阵列与另一个转录物的(部分)互补性引起的“特异性”可能导致信号与特定杂交体产生的信号不可区分。这种现象只能在缺乏待检测RNA(RNA无效突变体或高效RNAi敲除)的对照样品中检测。由于这些对照样本在大多数情况下不易获得,因此我们建议在设计阶段(启动程序B之前)对探针序列进行针对宿主转录组的BLAST探针序列,以排除此类隐蔽目标。
  18. 如果一个已经测试过且经过验证的工作探针组导致这种低灵敏度杂交,原因可能是其中一个缓冲液中的核酸酶污染。在这种情况下,建议替换用于杂交的所有缓冲液。

食谱

  1. TE缓冲区
    10 mM Tris碱
    1 mM EDTA
    调整pH值至8.0
  2. 1x PBS
    137mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4 4/2 1.8mM KH 2 PO 4 4/2 调整pH值到7.4
    在RT
    存放
  3. 定影剂
    10 ml 16%(v / v)EM级多聚甲醛
    30毫升1x PBS
    无菌过滤器,带0.22μm过滤器
    在4°C储存
  4. PBT

    1%PBS中的0.1%Triton X-100 在RT
    存放

  5. 1.5倍PAGE加载缓冲区 ¾在1x PBS中的8 M尿素
    ¼6倍凝胶加载染料
    在RT
    存放
  6. 10倍TBE
    1 M Tris基地
    1 M硼酸
    20 mM EDTA
    在RT
    存放
  7. 15%PA - 8 M尿素储备(200毫升)
    75 ml 40%(v / v)丙烯酰胺/双溶液
    20毫升10倍TBE
    96克尿素
    使用dH 2 O
    最高可达200毫升 搅拌并加热(最高100°C)直至尿素完全溶解
    在室温下储存,与光线隔离
  8. 20x SSC
    3 M NaCl
    300 mM柠檬酸钠
    pH 7.0
    在RT
    存放
  9. 两次洗涤 - HYBEC
    2x SSC
    15%(v / v)碳酸亚乙酯
    1 mM EDTA
    0.1%Triton X-100
    在RT
    存放
  10. 2x全HYBEC
    2x SSC
    15%(v / v)碳酸亚乙酯
    1 mM EDTA
    50μg/ ml肝素
    100μg/ ml鲑鱼精子ssDNA
    0.1%Triton X-100
    在RT
    存放
  11. 80%TDE
    80%(v / v)2,2'-硫代二乙醇(Sigma-Aldrich)
    20%(v / v)1x PBS
    充分混合并在RT
    储存
  12. 裂解缓冲液
    50 mM Tris-HCl pH 7.0
    10 mM EDTA
    1%(v / v)SDS
    新鲜1毫米PMSF
    新鲜的cOmplete™小型无EDTA蛋白酶抑制剂*
    1:2,000新鲜RiboLock RNAse抑制剂
  13. 捕获杂交缓冲液
    50 mM Tris-HCl pH 7.0
    750 mM NaCl
    1 mM EDTA
    1%(v / v)SDS
    15%(v / v)碳酸亚乙酯
    新鲜1毫米PMSF
    新鲜的cOmplete™ mini无EDTA蛋白酶抑制剂*
    1:2,000新鲜RiboLock RNAse抑制剂
  14. 低盐洗涤缓冲液
    2x SSC
    0.5%(v / v)SDS
    新鲜1毫米PMSF
    新鲜的cOmplete™ mini无EDTA蛋白酶抑制剂*
  15. 高盐洗涤缓冲液
    750 mM NaCl
    30 mM柠檬酸钠pH 7.0
    0.5%(v / v)SDS
    新鲜1毫米PMSF
    新鲜的cOmplete™ mini无EDTA蛋白酶抑制剂*
  16. 洗脱缓冲液
    10 mM Tris-HCl pH 7.0
    1 mM EDTA
    在RT
    存放

*注意:根据制造商的说明添加。

致谢

这项工作是由EMBL资助的。该协议改编自Gaspar 等人,2017b。作者声明不存在相互冲突的利益冲突。

参考

  1. Femino,A.M.,Fay,F.S.,Fogarty,K.和Singer,R.H。(1998)。 可视化单个RNA转录本原位。 科学 280(5363):585-590。
  2. Gaspar,I.和Ephrussi,A。(2015)。 数量优势:定量单分子RNA检测分析。 Wiley Interdiscip Rev Dev Biol 4(2):135-150。
  3. Gaspar,I.和Ephrussi,A。(2017)。 来自发育果蝇的离体 ooplasm提取物卵母细胞进行定量TIRF显微镜分析。 Bio-protocol 7(13)。
  4. Gaspar,I.,Sysoev,V.,Komissarov,A。和Ephrussi,A。(2017a)。 一种RNA结合的非典型原肌球蛋白可以动态地向oskar mRNPs募集驱动蛋白-1。 EMBO J 36(3):319-333。
  5. Gaspar,I.,Wippich,F。和Ephrussi,A。(2017b)。 酶促生产单分子FISH和RNA捕获探针 RNA 23(10):1582-1591。
  6. Jambor,H.,Surendranath,V.,Kalinka,A.T.,Mejstrik,P.,Saalfeld,S.and Tomancak,P。(2015)。 系统成像揭示了果蝇发育过程中mRNAs的特征和变化。 / a> eLife 4:e05003
  7. Khong,A.,Matheny,T.,Jain,S.,Mitchell,S.F。,Wheeler,J.R。和Parker,R。(2017)。 应激颗粒转录组揭示了压力颗粒中mRNA积累的原理。 Mol Cell 68(4):808-820 e805。
  8. Little,S. C.,Sinsimer,K. S.,Lee,J. J.,Wieschaus,E. F.和Gavis,E. R.(2015)。 独立协调贩卖单个果蝇胚芽质粒mRNA。 Nat Cell Biol 17(5):558-568。
  9. Raj,A.,van den Bogaard,P.,Rifkin,S.A。,van Oudenaarden,A。和Tyagi,S。(2008)。 使用多个单标记探针对个体mRNA分子进行成像 Nat Methods, 5(10):877-879。
  10. Sage,D.,Donati,L.,Soulez,F.,Fortun,D.,Schmit,G.,Seitz,A.,Guiet,R.,Vonesch,C。和Unser,M.(2017)。 DeconvolutionLab2:解卷积显微镜方法的开源软件 - 生物学家的图像处理。 方法 115:28-41。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: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. DOI: 10.21769/BioProtoc.2750.
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