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Sep 2020
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A Novel Method to Map Small RNAs with High Resolution
一种新的高分辨率小RNA图谱绘制方法   

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Abstract

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers.


Graphic abstract:



Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.


Keywords: Small RNA (小RNA), sRNA (sRNA), RNA detection (RNA检测), DNA-PAINT (DNA-PAINT), In situ hybridization (原位杂交), Super-resolution (超分辨率), Single molecule (单分子), Microscopy (显微镜), LNA (LNA)

Background

Light diffraction limits restrict imaging fluorophores closer than ~200 nm (Hell, 2009). Several imaging technologies have been created to overcome this limitation by exploiting transient fluorophore ON and OFF states (blinking), including stimulated emission depletion (STED) (Blom and Widengren, 2017), photoactivated localization microscopy (PALM) (Shroff et al., 2008), structured illumination microscopy (SIM) (Heintzmann and Huser, 2017), and stochastic optical reconstruction microscopy (STORM) (Bates et al., 2013). STORM increases the imaging resolution to around 30 nm (Xu and Liu, 2019); however, it relies on the stochastic blinking of fluorophores. As a result, multiplexing is often limited by the choice of fluorophores with ideal photophysical properties, including high photon numbers, low duty cycles, and high survival fractions, such as Alexa Fluor 647 and Dyomics 654 (Dempsey et al., 2011). Moreover, image quantitation is often challenging due to the unpredictability of the blinking properties of the chosen fluorophores and photobleaching (Schnitzbauer et al., 2017; Huang et al., 2020).


DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) was invented for ultra-high, sub-5 nm spatial detection of target protein molecules (Jungmann et al., 2014; Schnitzbauer et al., 2017; Strauss and Jungmann, 2020). It utilizes the base-pairing nature of single-stranded DNA molecules to their reverse complement strands. In the DNA-PAINT design, an approximately 10 nucleotide (nt)-long docking strand is linked to a target molecule via a biotin-streptavidin bridge or DBCO-sulfo-NHS ester chemical reaction (Schnitzbauer et al., 2017). An imager strand, conjugated to a fluorophore, is perfused in during the imaging process. The melting temperature (Tm) of the imager and docking strands is designed to be ~15°C. As a result, the imager strand-docking strand pairs will constantly bind and unbind at room temperature, which contributes to the blinking events, similar to the other super-resolution microscopy imaging techniques. The predictability of the binding duration and unlimited docking-imager strand combinations make DNA-PAINT ideal for quantitation and multiplexing (Jungmann et al., 2016).


sRNA-PAINT was created based on DNA-PAINT to detect small RNAs (sRNAs) that are usually ~10 nm in size (Huang et al., 2020). Our method preserves the nanometer resolution, quantitation, and multiplexing advantages of DNA-PAINT. In addition, we have introduced efficient and specific detection of sRNA targets with locked nucleic acid (LNA) (McTigue et al., 2004) and probe immobilization with 1-ethyl-1-(3-dimethylaminopropyl) carbodiimide (EDC) (Pena et al., 2009). sRNA-PAINT can be used for the multiplexed detection of sRNAs, their precursor mRNAs, and proteins in the sRNA biogenesis pathway when employed in combination with DNA-PAINT for understanding the relative localization patterns of small RNAs and their biogenesis machinery.

Materials and Reagents

  1. 20-ml glass scintillation vials (Electron Microscopy Sciences, catalog number: 72632)

  2. 50-ml Falcon tubes (Thermo Fisher Scientific, Corning, catalog number: 14-432-22)

  3. 0.5 M ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0) (Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: BP2482-500)

  4. 1 M Tris-HCl solution (pH 8.0) (Thermo Fisher Scientific, catalog number: AAJ22638AP)

  5. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: RT15710) (store at 4°C)

  6. 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (store at -20°C with a nitrogen seal)

  7. 200 proof ethanol (Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: 64-17-5) (store in a flammable cabinet)

  8. Deionized formamide (Millipore Sigma, catalog number: S4117) (store at 4°C)

  9. Denhardt’s solution (50×) (Millipore Sigma, catalog number: D2532) (store at -20°C)

  10. Dextran sulfate sodium salt (Millipore Sigma, catalog number: 67578) (store at 4°C)

  11. Histoclear II (VWR, Electron Microscopy Sciences, catalog number: 101412-878)

  12. NaCl (Millipore Sigma, catalog number: NIST975A)

  13. Paraffin wax pellets (Electron Microscopy Sciences, Paraplast X-tra, catalog number: 19214)

  14. Saline-sodium citrate (SSC) buffer (20×) (Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: BP1325)

  15. Sodium phosphate (Millipore Sigma, catalog number: 324283)

  16. tRNA (Millipore Sigma, catalog number: R1753) (store at -20°C)

  17. Ethanol gradient solutions (see Recipes)

  18. PHEM buffer (2×) (see Recipes)

  19. Fixation buffer (see Recipes)

  20. Enzyme solution (see Recipes)

  21. Glycine solution (see Recipes)

  22. TE buffer (see Recipes)

  23. TE-protease solution (see Recipes)

  24. Dextran sulfate solution (50%) (see Recipes)

  25. Highly abundant sRNA (see Recipes)

  26. Hybridization salt (see Recipes)

  27. Hybridization buffer (see Recipes)

  28. TBS buffer (pH 7.5) (see Recipes)

  29. tRNA solution (100 mg/ml) (see Recipes)

  30. Buffer C (see Recipes)

  31. EDC buffer (see Recipes)

  32. EDC solution (see Recipes)

Equipment

  1. DH40iL culture dish incubation system (Warner Instruments, model: 640388)

  2. Dissecting microscope (Zeiss M2BIO Dissecting Microscope)

  3. Flat-bottomed containers (Pyrex storage, 6 cup-1.5L)

  4. Fluigent Aria perfusion (Fluigent, Parts no: CB_SY_AR_1)

  5. Forceps (Electron Microscopy Sciences, catalog number: 72997-20)

  6. Glass staining dishes (Electron Microscopy Sciences, catalog number: 71426-DL)

  7. Grace Bio-Labs HybriSlip hybridization covers (Millipore Sigma, catalog number: 726024)

  8. Heat incubator (Fisher Scientific Isotemp incubator)

  9. Heat block for 1.5-ml tubes (BioExpress, Mini Dry Bath, model number: AS-BSH200-471)

  10. Hybridization oven (UVP HB-1000 hybridizer, model number: 95-0030-01)

  11. Masterflex C/L peristaltic pump (60 RPM) (Cole-Parmer Instrument, model: 77120-62)

  12. Paraffin microtome (Microm, model: MRK0403643)

  13. Parafilm (Thermo Fisher Scientific, BemisTM, catalog number: PM998)

  14. Plastic wrap (Thermo Fisher Scientific, FisherbrandTM Clear Plastic Wrap, catalog number: 12-640)

  15. Quick-release magnetic chamber for 25-mm low-profile round coverslips (Warner Instruments, model: 641943)

  16. Razor blades (Thermo Fisher Scientific, FisherbrandTM Razor Blades, catalog number: 22-305654)

  17. Slide warmer (Thermo Fisher Scientific, FisherbrandTM Slide Drying Bench, catalog number: 11-474-470)

  18. Stainless-steel glass rack (Electron Microscopy Sciences, catalog number:71426-R)

  19. Superfrost glass slides (Thermo Fisher Scientific, FisherbrandTM Tissue Path SuperfrostTM Plus Gold Slides, catalog number: 15-188-48)

  20. Tissue embedding and processing cassettes (Electron Microscopy Sciences, catalog number: 70070)

  21. Vacuum bell jar (Jelo Tech, model number: F42400-2221 with a Nisshin vacuum gauge, model number: B53595)

  22. Vacuum pump (Welch, model number: K48ZZMEM31)

  23. ValveLink8.2 Perfusion System (AutoMate Scientific, Berkeley, CA)

  24. Watercolor paintbrushes (#0)

  25. Wide Spectral Band 600 ± 100 nm Gold Fiducials coverglass (600-100AuF; Hestzig LLC, Leesburg, VA)

  26. Single-molecule localization microscope: Zeiss Elyra PS.1 super-resolution microscope (Carl Zeiss, Lberkochen, Germany) or Andor Dragonfly (Oxford Instruments, Abingdon, United Kingdom).

Software

  1. ImageJ ThunderSTORM (https://zitmen.github.io/thunderstorm/) (Ovesny et al., 2014)

  2. Picasso (https://github.com/jungmannlab/picasso) (Schnitzbauer et al., 2017)

Procedure

  1. Sample preparation

    1. Dissect the samples using a dissecting microscope with forceps and razor blades. This step can be omitted for cell cultures.

    2. Prepare the fixation buffer immediately prior to dissection. Place the dissected samples in an adequate amount of fixation buffer in 20-ml glass scintillation vials at room temperature. The volume of the fixative needs to be at least 15 times the volume of the sample.

    3. For plant samples, vacuum-infiltrate the samples (without the vial cap) for 15 min at 0.1 mPa in a vacuum bell jar. Repeat this step at least 3 times until the samples completely sink to the bottom of the fixation buffer.

    4. Store the fixed samples overnight at 4°C in fixation buffer. Samples can be stored at 4°C in fixation buffer for up to one week before embedding.

    5. Wash off the fixation buffer by incubating the samples in PBS (1×) buffer 3 times for 20 min each.

    6. Dehydrate the samples by incubation in an ethanol series of 25%, 50%, 75%, and 100% for 30 min each.

    7. Incubate the samples in 75% ethanol/25% histoclear, 50% ethanol/50% histoclear, and 25% ethanol/75% histoclear solution for 1 h each.

    8. Wash the samples 3 times in 100% histoclear for 1 h each.

    9. Add 5 ml fresh histoclear to the sample and then add paraplast to fill the jar. Incubate at 58°C overnight.

    10. The next day, add fresh paraplast to fill the jar. Repeat this step every hour until the melted wax fills up the jar.

    11. Wash the sample with freshly melted wax every 3 h. Repeat this step 3 times.

    12. Transfer the paraffin-embedded sample to the processing cassettes in the right orientation for sectioning.

    13. Cool the samples at room temperature. Store embedded samples at 4°C. Samples can be stored for up to 6 months.

    14. Section the samples using a paraffin microtome. The suggested section thickness is 6-10 µm.

    15. Transfer 1-2 sections carefully to the center of a gold fiducial-labeled coverglass using a paintbrush.

    16. Completely dry the samples by leaving the coverglass at 37°C for 2 days.


  2. Probe design

    1. VARNISH (Vetting and Analysis of RNA for in situ Hybridization) probes are designed automatically on our sRNA-PAINT probe design website hosted at the Donald Danforth Plant Science Center server: https://wasabi.ddpsc.org/~apps/varnish/.

    2. Enter the sRNA target sequence in the “Paste your small RNA sequence” field. Input the desired salt concentration and hybridization temperature (or use the default settings).

    3. Enter or select the docking strand from the docking strand dropdown menu.

    4. Enter or select the linker sequence.

    5. The results of the probe design will be sent to you via email with a link for ordering.


  3. In situ hybridization and washes

    1. Deparaffinize the samples twice by incubation in 100% histoclear for 10 min each.

    2. Rehydrate the samples by immersing the slides in an ethanol series of 100%, 95%, 80%, 70%, 50%, 30%, 10% (vol/vol), and water for 1 min each.

    3. Incubate the slides in 250 ml protease solution at 37°C for 20 min.

    4. Neutralize the samples in 250 ml 0.2% glycine solution for 20 min at room temperature.

    5. Wash the slides twice in PBS (1×) buffer for 2 min each.

    6. Dehydrate the samples by immersing the slides in water and ethanol series of 10%, 30%, 50%, 70%, 80%, 95%, and 100% for 1 min each.

    7. Immerse the slides in 100% ethanol for an additional 1 min.

    8. Store the samples in fresh 100% ethanol at 4°C for at least 4 h. Slides can be stored at 4°C for 1 week.

    9. Add 1 µl 250 µM probe to 9 µl water and 10 µl formamide. Heat denature at 90°C for 3 min and immediately chill on ice.

    10. Add 80 µl hybridization buffer to the solution mix in Step C9. Mix gently by pipetting without causing bubbles.

    11. Apply 100 µl probe mix to the sample slide and cover with hybridization membrane. Alternatively, simply place the slide (sample side down) on a wet paper towel that has been covered with a layer of parafilm such that the sample side touches the parafilm (Figure 1). Avoid bubbles during this step.



      Figure 1. Assembly of the sRNA-PAINT hybridization chamber


    12. Place the slide in a moist chamber and seal with plastic wrap. A simple moist chamber can be made by placing a sheet of parafilm on top of a wet paper towel in a flat-bottomed container.

    13. Hybridize at 53°C overnight (or at least 16 h).

    14. Pre-heat 0.2× SSC buffer to the same hybridization temperature as in Step C13.

    15. After hybridization, wash the samples twice with 0.2× SSC for 1 h each.

    16. Wash the samples in TBS (1×) buffer for 5 min at room temperature.

    17. To immobilize the hybridized probes, incubate the slides twice in freshly prepared EDC buffer at room temperature for 10 min each.

    18. Immerse the slides in EDC solution for 1 h and 15 min at room temperature.

    19. Neutralize the slides in 0.2% glycine solution for 30 min.

    20. Wash the slides twice in TBS (1×) buffer for 10 min each.

    21. Store the slides at 4°C in TBS (1×) buffer until imaging.


  4. sRNA-PAINT imaging

    1. Secure one slide on the imaging perfusion chamber with the sample side facing up (Figure 2).



      Figure 2. Assembly of the sRNA-PAINT imaging system. A. System for buffer storage and perfusion. B. Multiple tubes connect the perfusion unit to the imaging chamber. C. Imaging chamber setup for buffer exchange during image acquisition.


    2. Start the perfusion system and adjust the perfusion rate so that at least 2 ml imager solution is perfused through the sample during a 20-min imaging acquisition time.

    3. Image the sample on a microscope capable of total internal fluorescence microscopy (TIRFM) fitted with a 63× or 100× objective that is compatible with TIRFM. Use a 100-200 ms camera exposure time and collect about 10,000-20,000 frames for each imager strand. Save the images in a format that can be opened with Bio-Formats.

    4. Switch the perfusion input to buffer C and wash off the imager strand.

    5. Image the sample under the same conditions as in Step D3 to collect a buffer control image.

    6. Switch the perfusion input to a non-specific imager strand.

    7. Image the sample under the same conditions as Step D3 to collect a non-specific imager strand control image.

Data analysis

  1. Super-resolution imaging construction (Figure 3) using the ImageJ plugin ThunderSTORM: open ImageJ → Plugins → ThunderSTORM → Run analysis. Adjust the conditions and click on “Preview” to make sure that all specific spots are detected without any background being selected (usually, the default setting works well for most images). Click “OK.” This will generate an image file and a .txt file with the locations of all spots. Save the image file and .txt file.



    Figure 3. sRNA-PAINT imaging of a 24-nt phasiRNA in a 1.5-mm maize anther. A. sRNA-PAINT specifically detects the target signal. Alexa Fluor 647 was conjugated to imager strands. sRNA-PAINT images were acquired at a 1024×1024 pixel size and processed using the ThunderStorm ImageJ plugin. The final images are displayed at a 1024×1024 pixel size using equal brightness and contrast. Scale bar = 10 µm for all images. B. sRNA-PAINT achieved nanometer resolution of small RNAs. Nuclei are stained with DAPI (blue). Small RNA localization is shown as red/white dots.


  2. Quantitation using the Picasso software: a detailed user manual for the Picasso software is described by Schnitzbauer et al. (2017). Briefly, open Picasso: Localize and choose a suitable “Min. Net Gradient” so that all the spots are detected. Run “Localize (Identify & fit).” This will generate a .hdf5 file, which will be used in Picasso: Render for unit number calculation. The “Min. Net Gradient” sets the threshold to distinguish signal vs. background and needs to be consistent for all images. For sRNA copy number calculation, the non-specific background binding sites (buffer control, non-specific imaging strand, and ideally, a non-specific VARNISH probe) need to be subtracted from the copy number for each sRNA (Figure 4). In the example used in this manuscript, we selected an 80-nm pixel size as the area of interest to match the size of cells in the meiocyte. The average background detected was 17.10 ± 7.14 copies for the buffer control (Figure 3A, last panel) and 15.59±5.52 for the non-specific imager strand (Figure 3A, mid panel). In the final calculation, 17.10 was subtracted from the copy number for each cell layer.



    Figure 4. Quantitation of sRNA-PAINT using the Picasso software. A. Spot localization using Picasso: Localize function. B. Rendering and quantitation of the number of unit calculation using Picasso: Render. Yellow circles show the selected quantitation areas as an example of how the quantitation step was carried out in pollen mother cells.

Notes

  1. Protease treatment: The protease concentration can be adjusted based on the signal intensity. Normally, a 20-min incubation is sufficient at the provided concentration (130 µg/ml). A longer incubation time and an increased protease concentration can be combined if low signal intensity is observed.

  2. Probe concentration: Normally, 10 µM is sufficient for most sRNA detections. A probe concentration series can also be performed with 1 µM, 10 µM, and 100 µM to determine the most suitable concentration for sRNA targets.

  3. Hybridization temperature: Generally, 50-60°C is suitable for most VARNISH probes that are designed with a TM above 80°C. The ideal hybridization temperature can be determined by the melting temperature (Tm) of each VARNISH probe.

  4. EDC treatment: EDC treatment is used to attach the 5’ end of the VARNISH probes to the sample. EDC treatment has been shown to decrease VARNISH probe wash-off during multiplexed exchange-PAINT.

  5. Imager strand concentration: Imager strand concentration should be determined experimentally. Start with a lower concentration around 0.5 nM and gradually increase the imager strand until a good blinking rate (15-30 molecules per frame) is reached for each imaging frame.

  6. Choices of imager strands: Imager strands chosen for each sample should not interfere with the sample nonspecifically. To test imager strand (non-) specificity, add 2 nM each imager strand to the sample before hybridization and image under the same conditions that will be used in later image acquisition steps. Only imager strands that do not interfere with samples should be used in subsequent experiments.

  7. Perfusion system: The perfusion systems that we have tested are the Fluigent Aria perfusion unit using air pressure, the ValveLink8.2 perfusion system using gravity flow, and a syringe pump system using syringe pressure force. The Fluigent system works best for multiplexed sRNA-PAINT, but it is costly. The syringe pump system is convenient and cost-efficient, and is an ideal starting unit for 1-2 imager strands.

Recipes

  1. Ethanol gradient solutions

    To generate 10%, 30%, 50%, 70%, 80%, 95%, and 100% ethanol series, mix 50 ml, 150 ml, 250 ml, 350 ml, 400 ml, 475 ml, and 500 ml ethanol and bring to 500 ml with nanopure water.

  2. PHEM buffer (2×)

    1. Add 18.14 g PIPES, 6.5 g HEPES, 3.8 g EGTA, and 0.99 g MgSO4 to 400 ml water and bring to a final volume of 500 ml. The final concentration of each ingredient is: 60 mM PIPES, 5 mM HEPES, 10 mM EGTA, 2 mM MgSO4, pH 8.

    2. Autoclave and store the solution at 4°C.

  3. Fixation buffer

    1. Add 10 ml 16% paraformaldehyde and 20 ml 2× PHEM buffer to a 50-ml tube.

    2. Add water to bring up to 50 ml. This step should be carried out in a fume hood. Prepare immediately before use.

  4. Enzyme solution

    1. Dissolve 0.5 g protease in 10 ml water.

    2. Pre-digest the solution at 37°C for 4 h. The final stock concentration is 50 mg/ml.

    3. Store 650-µl aliquots at -20°C.

  5. Glycine solution

    1. Add 5 ml 10% (w/v) glycine solution to 245 ml PBS buffer (1×). The final concentration of glycine solution is 0.2% (w/v).

    2. Filter sterilize and store the solution at 4°C.

  6. TE buffer

    Add 100 ml 100 mM Tris-HCl and 100 ml 10 mM ethylenediaminetetraacetic acid (EDTA) to 800 ml water. Store at room temperature.

  7. TE-protease solution

    Add 650 µl enzyme solution to 250 ml TE buffer. Warm to 37°C. Prepare immediately before use.

  8. Dextran sulfate solution (50%)

    1. Add 5 g dextran sulfate to 7 ml water.

    2. Dissolve dextran sulfate using 80°C heat.

    3. Bring up to 10 ml with water.

    4. Aliquot to 2.5 ml and store at -20°C.

  9. Highly abundant sRNA

    A highly abundant sRNA with a specific localization pattern can be used as a positive control. For example, miR2275 has been used as a positive control for sRNA-PAINT in maize anthers in our previous experiments due to its high abundance during meiosis and specific accumulation in the tapetal cell layer in meiotic anthers.

  10. Hybridization salt

    1. Add 1.75 g NaCl and 164 mg sodium monophosphate to 1 ml Tris-HCl (pH 8.0) and 1 ml 0.5 M EDTA.

    2. Bring up to 10 ml with water.

    3. Store 1.25-ml aliquots at -20°C.

  11. Hybridization buffer

    1. Add 1.25 ml hybridization salts, 5 ml deionized formamide, 2.5 ml 50% dextran sulfate, 250 μl 50× Denhardt’s solution, and 125 μl tRNA solution to 875 μl water.

    2. Filter-sterilize and store 1-ml aliquots at -20°C.

  12. TBS buffer (pH 7.5)

    1. Add 100 ml 500 mM Tris-HCl and 30 ml 5 M NaCl to water.

    2. Bring up to 1 L.

    3. TBS is stable at 4°C for 3 months.

  13. tRNA solution (100 mg/ml)

    Add 1 g tRNA to 1 ml water. Mix and store at -20°C.

  14. Buffer C

    Add 100 ml 5 M NaCl to 900 ml PBS buffer (1×).

  15. EDC buffer

    1. Add 400 µl 1-methylimidazole, 112.5 µl 12M HCl, and 2.4 ml 5 M NaCl to 30 ml water.

    2. Bring up to 40 ml with water.

    3. Prepare immediately before use.

  16. EDC solution

    Add 0.31 g EDC to 40 ml EDC buffer.

Acknowledgments

This project was supported by the US NSF Plant Genome Research Program (awards 1649424, 1611853, and 1754097). We thank the members of the Meyers and Caplan labs for their support, and Joanna Friesner for editorial assistance. We thank Virginia Walbot (Stanford University) and the members of her lab for supplying maize materials and for useful discussions on anther small RNAs. We thank Prof. Ralf Jungmann for his support in DNA-PAINT, qPAINT, and analysis using Picasso. Microscopy equipment was acquired with a shared instrumentation grant (S10 OD016361), and access was supported by the NIH-NIGMS (P20 GM103446), the NSF (IIA-1301765), and the State of Delaware. The original research paper that developed sRNA-PAINT is published in Nucleic Acids Research: doi.org/10.1093/nar/gkaa623.

Competing interests

No competing interests.

References

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  3. Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. and Zhuang, X. (2011). Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8(12): 1027-1036.
  4. Heintzmann, R. and Huser, T. (2017). Super-Resolution Structured Illumination Microscopy. Chem Rev 117(23): 13890-13908.
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  8. Jungmann, R., Avendano, M. S., Woehrstein, J. B., Dai, M., Shih, W. M. and Yin, P. (2014). Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11(3): 313-318.
  9. McTigue, P. M., Peterson, R. J. and Kahn, J. D. (2004). Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry 43(18): 5388-5405.
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  11. Pena, J. T., Sohn-Lee, C., Rouhanifard, S. H., Ludwig, J., Hafner, M., Mihailovic, A., Lim, C., Holoch, D., Berninger, P., Zavolan, M. and Tuschl, T. (2009). miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6(2): 139-141.
  12. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. and Jungmann, R. (2017). Super-resolution microscopy with DNA-PAINT. Nat Protoc 12(6): 1198-1228.
  13. Shroff, H., Galbraith, C. G., Galbraith, J. A. and Betzig, E. (2008). Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5(5): 417-423.
  14. Strauss, S. and Jungmann, R. (2020). Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT. Nat Methods 17(8): 789-791.
  15. Xu, J. and Liu, Y. (2019). Imaging Higher-order Chromatin Structures in Single Cells Using Stochastic Optical Reconstruction Microscopy. Bio-protocol 9(3): e3160.

简介

[摘要]分析细胞结构和分子的相对位置对于解决生物学问题至关重要。绕过光衍射极限的超分辨率显微镜技术在原位研究细胞分子动力学方面变得越来越流行。然而,所述的超分辨率成像技术应用到检测小RNA(的sRNA)由适当的荧光团,样品的自发荧光,以及未能多路复用的选择的限制。在这里,我们描述了一个斯尔纳-PAINT协议的检测的sRNA的以纳米分辨率。该方法结合锁核酸探针的特异性和低的背景,精确孔定量吨通货膨胀和DNA点积累用于显像纳米形貌(DNA-PAINT)的复用的特征。使用这种方法,我们成功定位,它们对玉米的花药发展具有重要意义,在亚20nm的分辨率和孔定量斯尔纳目标达ED其确切的拷贝数。

图文摘要:

多重 sRNA-PAINT。具有不同对接链(即,a、b、...)的原位杂交 (VARNISH) 探针的 RNA 的多重审查和分析将与样品杂交。第一个探头将使用 a* 成像器进行成像。a* 成像器将被缓冲液 C 洗掉,然后样品将被 b* 成像器成像。可以按顺序重复洗涤和成像步骤以进行多路复用。

[背景]光衍射极限限制成像荧光团接近约 200 nm (Hell, 2009) 。已经创建了几种成像技术来通过利用瞬态荧光团 ON 和 OFF 状态(闪烁)来克服这一限制,包括受激发射耗尽 (STED)(Blom 和 Widengren,2017 年)、光激活定位显微镜(PALM)(Shroff等人,2008 年) )、结构照明显微镜 (SIM)(Heintzmann 和 Huser,2017 年)和随机光学重建显微镜(STORM)(Bates等人,2013 年)。STORM 将成像分辨率提高到 30 nm 左右(Xu 和 Liu,2019);然而,它依赖于荧光团的随机闪烁。因此,多路复用通常受限于选择具有理想光物理特性的荧光团,包括高光子数、低占空比和高存活率,例如 Alexa Fluor 647 和 Dyomics 654 (Dempsey et al. , 2011)。此外,图像孔定量吨通货膨胀通常具有挑战性,由于不可预测的所选择的荧光团的闪烁特性和光漂白(Schnitzbauer等人,2017 ;黄。等人,202 0 )。

用于纳米级地形成像的 DNA 点积累 (DNA-PAINT) 被发明用于对目标蛋白质分子进行超高、亚 5 nm 空间检测(Jungmann等人,2014 年;Schnitzbauer等人,2017 年;Strauss 和 Jungmann,2020 年) )。它利用单链 DNA 分子与其反向互补链s的碱基配对特性。在DNA-PAINT设计中,约10个核苷酸(nt)-长对接链通过生物素-链霉亲和桥或DBCO磺基-NHS酯化学反应连接到靶分子(Schnitzbauer等人,2017)。在成像过程中灌注与荧光团结合的成像链。成像器和对接链的熔解温度 (T m ) 设计为~15°C。因此,成像器链对接链对将在室温下不断结合和解绑,这与其他超分辨率显微成像技术类似,这会导致闪烁事件。结合持续时间和无限对接成像器链的组合的可预测性使DNA-PAINT理想的孔定量吨通货膨胀和复用(容曼等人,2016)。

sRNA-PAINT 是基于 DNA-PAINT 创建的,用于检测大小通常约为 10 nm 的小 RNA (sRNA)(Huang等人,2020 年)。我们的方法保留了纳米级分辨率,孔定量吨通货膨胀,和DNA-PAINT的复用优点。此外,我们还引入了使用锁核酸 (LNA)(McTigue等人,2004 年)和 1-乙基-1-(3-二甲氨基丙基)碳二亚胺 (EDC) 固定探针的高效和特异性检测 sRNA 目标(Pena等人等,2009)。的sRNA-PAINT可以使用用于当多路检测的sRNA的,其前体的mRNA和蛋白质在生物发生的sRNA途径使用结合DNA-PAINT理解的小RNA的相对定位模式和它们的生物合成机器。

关键字:小RNA, sRNA, RNA检测, DNA-PAINT, 原位杂交, 超分辨率, 单分子, 显微镜, LNA

材料和试剂
 
20 - ml 玻璃闪烁瓶(Electron Microscopy Sciences,目录号:72632)
50 - ml Falcon 管(Thermo Fisher Scientific,Corning,目录号:14-432-22)
的0.5M Ë thylenediaminetetraacetic一个CID(EDTA)溶液(pH 8.0)(赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:BP2482-500)
1 M Tris-HCl溶液(pH 8.0)(Thermo Fisher Scientific,目录号:AAJ22638AP)
16% 多聚甲醛(Electron Microscopy Sciences,目录号:RT15710)(在 4°C 下储存)
1 -乙基- 3 - (3 -二甲基氨基丙基)碳二亚胺(EDC)(在-20℃下存储与一个氮气密封)
200标准乙醇(赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:64-17-5)(以存储一个易燃柜)
去离子甲酰胺(Millipore Sigma,目录号:S4117)(在4°C下储存)
Denhardt 的解决方案 (50×)(Millipore Sigma,目录号:D2532)(储存在-20 °C)
硫酸葡聚糖钠盐(Millipore Sigma,目录号:67578)(在4 °C下储存)
Histoclear II(VWR,Electron Microscopy Sciences,目录号:101412-878)
NaCl(Millipore Sigma,目录号:NIST975A)
石蜡颗粒(Electron Microscopy Sciences,Paraplast X-tra,目录号:19214)
盐水 - 柠檬酸钠(SSC)缓冲液(20 × )(Thermo Fisher Scientific,Fisher BioReagents TM ,目录号:BP1325)
磷酸钠(Millipore Sigma,目录号:324283)
tRNA(Millipore Sigma,目录号:R1753)(储存在-20°C)
乙醇梯度溶液(见配方)
PHEM 缓冲液(2 × ) (见配方)
固定缓冲液(见配方)
酶溶液(见配方)
甘氨酸溶液(见配方)
TE 缓冲液(见配方)
TE-蛋白酶溶液(见配方)
硫酸葡聚糖溶液(50%)(见配方)
高丰度的 sRNA (见食谱)
杂交盐(见配方)
杂交缓冲液(见配方)
TBS 缓冲液(pH 7.5)(见配方)
tRNA 溶液(100 毫克/毫升)(见食谱)
缓冲液 C (见配方)
EDC 缓冲液(见配方)
EDC 解决方案(见配方)
 
设备
 
DH40iL培养皿incubat离子系统(华纳仪器,型号:640388)
解剖显微镜(蔡司 M2BIO 解剖显微镜)
平底ed容器s (耐热玻璃储存,6 杯-1.5 升)
Fluigent Aria 灌注(Fluigent,部件编号:CB_SY_AR_1)
镊子(电子显微镜科学,目录号:72997-20)
玻璃染色皿(电子显微镜科学,目录号:71426-DL)
格雷斯生物实验室HybriSlip杂交盖小号(Millipore公司Sigma,目录号:726024)
加热培养箱(Fisher Scientific Isotemp 培养箱)
热b锁1.5 -毫升管(BioExpress,迷你干澡,型号:AS-BSH200-471)
杂交炉(UVP HB-1000 杂交仪,型号:95-0030-01)
Masterflex C/L 蠕动泵 (60 RPM)(Cole-Parmer 仪器,型号:77120-62)
石蜡切片机(Microm,型号:MRK0403643)
Parafilm(Thermo Fisher Scientific,Bemis TM ,目录号:PM998)
保鲜膜(Thermo Fisher Scientific,Fisherbrand TM Clear Plastic Wrap,目录号:12-640)
快速-释放磁性室25 -毫米低-剖面圆形盖玻片(华纳仪器,型号:641943)
剃刀刀片(Thermo Fisher Scientific,Fisherbrand TM Razor Blades,目录号:22-305654)
玻片加热器(Thermo Fisher Scientific,Fisherbrand TM Slide Drying Bench,目录号:11-474-470)
不锈钢-玻璃钢机架(电子显微镜科学,目录号:71426-R)
Superfrost 载玻片(Thermo Fisher Scientific,Fisherbrand TM Tissue Path Superfrost TM Plus Gold Slides,目录号:15-188-48)
组织包埋和处理盒(电子显微镜科学,目录号:70070)
真空钟罩(Jelo Tech,型号:F42400-2221,带 Nisshin 真空计,型号:B53595)
真空泵(韦尔奇,型号:K48ZZMEM31)
ValveLink8.2 灌注系统(AutoMate Scientific,加州伯克利)
水彩画笔 (#0)
宽光谱带 600 ± 100 nm Gold Fiducials coverglass (600-100AuF; Hestzig LLC, Leesburg, VA)
单-分子定位显微镜:蔡司Elyra PS.1超分辨率显微镜(Carl Zeiss,Lberkochen,德国)或安道尔蜻蜓(牛津仪器,阿宾登,英国)。
 
软件
 
ImageJ ThunderSTORM ( https://zitmen.github.io/thunderstorm/ ) ( Ovesny et al. , 2014)。
毕加索(https://github.com/jungmannlab/picasso)(Schnitzbauer等人,2017) 。
 
程序
 
样品制备
解剖的使用具有钳子和刀片在解剖显微镜的样品。对于细胞培养可以省略此步骤。
制备的固定缓冲器立即之前到dissecti上。放置在在固定缓冲液的足够量的切割样品在20 -毫升玻璃闪烁瓶中在室温。固定剂需要的体积小号是样品的至少15倍的体积。
对于植物样品,真空-渗透的样品(不含所述瓶盖)在真空钟罩15分钟,0.1毫帕。重复此步骤,至少3次,直到所述样品完全沉到固定缓冲液的底部。
存储在4℃过夜固定的样品°的固定缓冲液C。样品可以贮存于4℃下在固定缓冲液为至多一个嵌入前一周。
通过孵育洗掉固定缓冲液的样品在PBS(1 × )缓冲液3次对每20分钟。
脱水的样品通过在孵育的乙醇系列的25%,50%,75%和100%为30分钟的每个。
将样品在 75% 乙醇/25% histoclear、50% 乙醇/50% histoclear和25% 乙醇/75% histoclear 溶液中孵育1 小时。
洗样品3次,在100%histoclear为每1个小时。
将 5 ml 新鲜的 histoclear 添加到样品中,然后添加 paraplast 以填充罐子。在 58°C 下孵育过夜。
第二天,添加新鲜的 paraplast 以填充罐子。重复此步骤,每一个小时未直到融化的蜡填满了瓶子里。
每 3 小时用刚融化的蜡清洗样品。重复此步骤 3 次。
将石蜡包埋的样品以正确的方向转移到处理盒中进行切片。
在室温下冷却样品。将嵌入的样品储存在 4°C。样品可以保存长达 6 个月。
部分的样品用石蜡切片。在S uggested部厚度是6 - 10微米。
使用画笔将 1-2 部分小心地转移到金色基准标记的玻璃盖板的中心。
将盖玻片在 37°C 下放置 2 天,使样品完全干燥。
 
探头设计
VARNISH(原位杂交RNA 的审查和分析)探针在我们的 sRNA-PAINT 探针设计网站上自动设计,该网站位于 Donald Danforth 植物科学中心服务器:https : //wasabi.ddpsc.org/~apps/varnish/。
在“粘贴您的小 RNA 序列”字段中输入 sRNA 目标序列。输入的期望的盐的浓度和杂交温度(或使用的默认设置)。
从对接链下拉菜单中输入或选择对接链。
输入或选择链接器序列。
结果中的探头设计将通过电子邮件的链接订货发送给您。
 
原位杂交和洗涤
Deparaffinize的样品两次孵育在100%histoclear对于每次10分钟。
再水化的浸渍样品的在幻灯片的乙醇系列的100%,95%,80%,70%,50%,30%,10%(体积/体积),和水对于每1分钟。
将载玻片在 250 ml 蛋白酶溶液中在 37 °C 下孵育20 分钟。
中和的250毫升0.2%的甘氨酸溶液样品,在室温下20分钟。
洗的载玻片两次P中BS(1 × )缓冲液对各2分钟。
脱水的浸渍样品的在水滑梯和乙醇系列的10%,30%,50%,70%,80%,95%,和100%对每1分钟。
浸入的在100%乙醇玻片一个附加1分钟。
将样品在 4°C 的新鲜 100% 乙醇中储存至少 4 小时。载玻片可在 4°C 下保存 1 周。
将 1 µl 250 µM 探针添加到 9 µl 水和 10 µl 甲酰胺中。在 90 °C 下加热变性3 分钟,然后立即在冰上冷却。
添加80μl的杂交缓冲液中,以在该溶液中混合小号TEP Ç 9.轻轻混合通过而不引起气泡吸移。
将 100 µl 探针混合物涂在样品载玻片上并盖上杂交膜。备选地,简单地放置在已经覆盖用石蜡膜的层的湿纸巾滑动(样品面朝下),例如使样品侧接触石蜡膜(图1 )。在此步骤中避免气泡。
 
 
图1 。组件的所述的sRNA-PAINT杂交室
 
将载玻片放在潮湿的室内,并用塑料薄膜密封。一个简单的保湿室中可以通过进行在平底放置封口膜的片材上的湿纸巾的顶版的容器。 
在 53 °C 下杂交过夜(或至少 16 小时)。
预热0.2 × SSC缓冲液,以同样的杂交温度在小号TEP Ç 13。
杂交后,洗涤样品两次用0.2 × SSC为每1个小时。
在室温下在 TBS (1 × ) 缓冲液中清洗样品5 分钟。
以固定杂交的探针,孵育载玻片在新鲜制备的EDC缓冲液洗涤两次,在室温下为每10分钟。
在室温下将幻灯片浸入 EDC 溶液中 1 小时 15 分钟。
在 0.2% 甘氨酸溶液中中和幻灯片 30 分钟。
洗载玻片两次在TBS(1 × )缓冲液对各10分钟。
存储的小号在大环内酯类4 ℃下在TBS(1 × )缓冲液,直到成像。
 
sRNA-PAINT成像
将一张载玻片固定在成像灌注室上,样品侧朝上(图2 )。
 
 
图2 。组件的所述的sRNA-PAINT成像系统。A. Ş ystem的缓冲存储器和灌注。B. 多个管将灌注单元连接到成像室。C. 图像采集过程中缓冲液交换的成像室设置。
 
开始灌注系统和调节灌注速率,使得至少2毫升成像器溶液中20通过样品灌注-分钟成像采集时间。
在配备与 TIRFM 兼容的 63 ×或 100 ×物镜的全内部荧光显微镜 (TIRFM) 显微镜上对样品进行成像。使用100-200毫秒相机的曝光时间,并收集大约10,000 -对于每个成像股20000帧。保存在中能与生物格式打开的格式的图像。
将灌注输入切换到缓冲区 C 并洗掉成像器链。
图像中的采样下相同的条件š在小号TEP d 3来收集缓冲器控制图像。
将灌注输入切换到非特定成像器链。
图像相同的条件下的样品小号作为步骤D3以收集非特异性成像器链的控制图像。
 
数据分析
 
超分辨率成像结构(图3使用)的ImageJ的插件雷暴:开放的ImageJ 插件雷暴运行分析。调整的条件,点击“预览”,以确保作出这样的不被选择的背景检测到所有的特定点(通常是默认设置适用于大多数图像)。单击“确定” 。”这将生成一个图像文件和 .txt 文件,其中包含所有点的位置。保存图像文件和 .txt 文件。
 
 
图3 。的sRNA在24 nt的phasiRNA的-PAINT成像一个1.5 -毫米玉米花药。A. sRNA-PAINT 专门检测目标信号。Alexa Fluor 647 与成像链偶联。的sRNA-PAINT图像是在获取一个1024 × 1024像素尺寸和使用处理的雷暴ImageJ的插件。最终图像一个重新显示在一个1024 × 1024像素尺寸使用等于亮度和对比度。所有图像的比例尺 = 10 µm。B. sRNA-PAINT 实现了小 RNA 的纳米分辨率。核一用DAPI(蓝色)染色重。小RNA本地化一个示出重新作为红色/白色点。
 
孔定量吨通货膨胀使用的毕加索软件:一个用于详细的用户手册的毕加索软件是由所述(Schnitzbauer等人,2017)。简而言之,打开 Picasso: Localize 并选择合适的“Min. 净梯度”,让所有的检测点。运行“Localize (Identify & fit)” 。” 这将生成一个.hdf5 文件,该文件将在 Picasso: Render 中用于单元数计算。“闵。净梯度”设置Ş区分信号与背景和需求是所有的图片一致的门槛。对于 sRNA 拷贝数计算,需要从每个 sRNA 的拷贝数中减去非特异性背景结合位点(缓冲液控制、非特异性成像链,理想情况下是非特异性 VARNIH 探针)(图4 )。在该手稿中使用的例子中,我们选择了一个80 -纳米像素大小作为所述感兴趣的区域,以在匹配性母细胞的细胞的尺寸。该平均背景检测为17.10±7.14 COP IES为的缓冲控制(图3 A,最后面板)和15.59±5.52对的非特异性成像器链(图3 A,中间面板)。在最后的计算,17.10从拷贝数中减去对于各细胞层。
 
 
图4 。孔定量牛逼使用斯尔纳-PAINT的通货膨胀的毕加索软件。A.使用Picasso 进行现场定位:Localize 功能。B.渲染和孔定量吨单位计算的数目的通货膨胀使用毕加索:渲染。黄色圆圈表示的选择的孔定量吨通货膨胀领域的孔定量如何一例吨通货膨胀步骤进行花粉母细胞。
 
笔记
 
蛋白酶处理:所述蛋白酶浓度可以根据被调节上的信号强度。通常情况下,一个20 -分钟的温育是在足以将提供浓度(130微克/毫升)。甲升onger孵育时间和一个增加的蛋白酶浓度可以如果观察低信号强度相结合。
探针浓度:通常,对于大多数 sRNA 检测,10 µM 就足够了。还可以使用 1 µM、10 µM 和 100 µM执行探针浓度系列,以确定最适合 sRNA 目标的浓度。
杂交温度:一般来说,50 - 60°C 适合大多数设计有80°C 以上 TM 的VARNISH 探针。i deal 杂交温度可以通过每个 VARNIH 探针的解链温度 (T m )来确定。
EDC 处理:EDC 处理用于将 VARNISH 探针的 5' 端连接到样品上。EDC治疗已经显示出减少VARNISH探针洗涤-断多路复用交换-PAINT期间。
成像链浓度:成像链浓度应通过实验确定。开始与周围为0.5nM浓度较低,并逐渐增加成像器链,直至良好的闪烁速率(15 -每帧30分子)达到用于每个成像帧。
成像链的选择:为每个样品选择的成像链不应非特异性干扰样品。为了测试成像器链(非- )特异性,杂交和图像之前添加2nM的每个成像器链的样品下,将用于在相同的条件在后面的图像采集步骤。在随后的实验中只应使用不干扰样品的成像链。
灌注系统:在灌注系统的是我们已经测试是使用空气压力的Fluigent咏叹调灌注单元,所述ValveLink8.2 p erfusion小号ystem使用重力流,并且使用注射器压力的注射器泵系统。该Fluigent系统最适合多路复用斯尔纳-PAINT,但它是昂贵的。注射泵系统方便和成本有效的,并且是开始单元1-2成像器链的理想。
 
食谱
 
乙醇梯度溶液
要生成 10%、30%、50%、70%、80%、95% 和 100% 乙醇系列,请混合 50 ml、150 ml、250 ml、350 ml、400 ml 、475 ml 和 500 ml 乙醇并用纳米纯水定容至 500 ml。
PHEM 缓冲液(2 × )
将18.14 g PIPES、6.5 g HEPES、3.8 g EGTA 和 0.99 g MgSO 4 添加到 400 ml 水中,并使其最终体积为 500 ml。最终浓度的各成分为:60mM的PIPES,5mM的HEPES,10mM的EGTA,2mM的硫酸镁4 ,pH为8。
高压釜中,存储了在4℃下溶液。
固定缓冲液
甲DD10毫升16%多聚甲醛和20ml的2- × PHEM缓冲到一个50 -毫升管。
加水至 50 毫升。此步骤应在通风橱中进行。使用前立即准备。
酶液
D将 0.5 g 蛋白酶溶解在 10 ml 水中。
在 37 °C 下预消化溶液4 小时。在F伊纳勒库存浓度为50毫克/毫升。
在 -20°C 下储存 650 - µl 等分试样。
甘氨酸溶液
将5 ml 10% (w/v) 甘氨酸溶液加入 245 ml PBS 缓冲液 (1 × )。在F的甘氨酸溶液伊纳勒浓度为0.2%(W / V)。
过滤消毒并保存了在4℃下溶液。
TE缓冲液
将100 ml 100 mM Tris-HCl 和 100 ml 10 mM 乙二胺四乙酸 (EDTA) 添加到 800 ml 水中。在室温下储存。
TE-蛋白酶溶液
将650 µl 酶溶液添加到 250 ml TE 缓冲液中。加热至 37°C。使用前立即准备。
硫酸葡聚糖溶液 (50%)
将5 克硫酸葡聚糖加入 7 毫升水中。
使用 80°C 加热溶解硫酸葡聚糖。
带来最多至10ml水。
分装至 2.5 ml 并储存在 -20°C。
高丰度sRNA
高度丰富的sRNA与一个特定定位模式可以用来作为阳性对照。例如,在我们之前的实验中,miR2275 已被用作玉米花药中 sRNA-PAINT的阳性对照,因为它在减数分裂期间的高丰度和在减数分裂花药的绒毡层细胞层中的特异性积累。
杂交盐
将1.75 g NaCl 和 164 mg 单磷酸钠加入 1 ml Tris-HCl (pH 8.0) 和 1 ml 0.5 M EDTA。
加水至 10 毫升。
在 -20°C 下储存 1.25 - ml 等分试样。
杂交缓冲液
将1.25 ml 杂交盐、5 ml 去离子甲酰胺、2.5 ml 50% 硫酸葡聚糖、250 μl 50 × Denhardt 溶液和 125 μl tRNA 溶液添加到 875 μl 水中。
过滤灭菌并在 -20°C 下储存 1 - ml 等分试样。
TBS 缓冲液(pH 7.5)
将100 ml 500 mM Tris-HCl 和 30 ml 5 M NaCl 添加到水中。
最多 1 L。
TBS 在 4°C 下可稳定保存 3 个月。
tRNA 溶液(100 毫克/毫升)
将1 g tRNA 添加到 1 ml 水中。混合并储存在 -20°C。
缓冲液 C
添加100 ml 5 M NaCl 到 900 ml PBS 缓冲液 (1 × )。
EDC缓冲器
甲DD400μl的1 -甲基咪唑,112.5微升12M的HCl,和2.4毫升5 M氯化钠至30毫升水中。
带来了至40ml水。
使用前立即准备。
EDC解决方案
将0.31 g EDC 添加到 40 ml EDC 缓冲液中。
 
致谢
 
该项目由美国国家科学基金会植物基因组研究计划资助(奖励1649424,1611853 ,和1754097 )。我们感谢在了迈尔斯和卡普兰实验室的成员的支持,和Joanna Friesner为编辑协助。我们感谢弗吉尼亚·沃尔伯特(斯坦福大学),并在她的实验室的供应玉米材料和花药小RNA有益的讨论成员。我们感谢拉尔夫·容曼教授为他的DNA-PAINT,Q漆的支持,以及分析使用毕加索。显微镜设备与共享仪器补助金(S10 OD016361)和访问获得由美国国立卫生研究院NIGMS(P20 GM103446),美国国家科学基金会(IIA-1301765)的支持,和特拉华州。最初的研究论文是开发编斯尔纳-PAINT发表在核酸研究:doi.org/10.1093/nar/gkaa623。
 
利益争夺
 
没有竞争的利益。
 
参考
 
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Huang, K., Demirci, F., Batish, M., Treible, W., Meyers, BC 和 Caplan, JL (2020)。使用 sRNA-PAINT 对小 RNA 进行定量、超分辨率定位。核酸研究48(16):e96。
Jungmann, R., Avendano, MS, Dai, M., Woehrstein, JB, Agasti, SS, Feiger, Z., Rodal, A. 和 Yin, P. (2016)。使用 qPAINT 进行定量超分辨率成像。Nat 方法13(5): 439-442。
Jungmann, R., Avendano, MS, Woehrstein, JB, Dai, M., Shih, WM 和 Yin, P. (2014)。使用 DNA-PAINT 和 Exchange-PAINT 进行多重 3D 细胞超分辨率成像。Nat 方法11(3): 313-318。
McTigue, PM、Peterson, RJ 和 Kahn, JD (2004)。锁核酸 (LNA)-DNA 双链体形成的序列相关热力学参数。生物化学43(18):5388-5405。
Ovesny, M.、Krizek, P.、Borkovec, J.、Svindrych, Z. 和 Hagen, GM (2014)。ThunderSTORM:用于 PALM 和 STORM 数据分析和超分辨率成像的综合 ImageJ 插件。生物信息学30(16):2389-2390。
Pena, JT, Sohn-Lee, C., Rouhanifard, SH, Ludwig, J., Hafner, M., Mihailovic, A., Lim, C., Holoch, D., Berninger, P., Zavolan, M. 和Tuschl, T. (2009)。甲醛和 EDC 固定组织中的 miRNA 原位杂交。Nat 方法6(2): 139-141。              
Schnitzbauer, J.、Strauss, MT、Schlichthaerle, T.、Schueder, F. 和 Jungmann, R.(2017 年)。使用 DNA-PAINT 的超分辨率显微镜。国家议定书 12(6): 1198-1228。              
Shroff, H.、Galbraith, CG、Galbraith, JA 和 Betzig, E.(2008 年)。纳米级粘附动力学的活细胞光活化定位显微镜。Nat 方法5(5): 417-423。              
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引用:Huang, K., Demirci, F., Meyers, B. C. and Caplan, J. L. (2021). A Novel Method to Map Small RNAs with High Resolution. Bio-protocol 11(16): e4128. DOI: 10.21769/BioProtoc.4128.
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