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Single-probe RNA FISH in Yeast
酵母菌单探针 RNA FISH   

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Nucleic Acids Research
Sep 2017

Abstract

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

Keywords: RNA FISH (RNA FISH), Fluorescence In Situ Hybridization (荧光原位杂交), Saccharomyces cerevisiae (酿酒酵母), Budding yeast (芽殖酵母), Transcription (转录), Single molecule (单分子)

Background

Precise quantification of the transcript profile of single cells is possible by single molecule Fluorescence In Situ Hybridization (smFISH). This procedure gives good signal to noise by targeting a single mRNA molecule with multiple fluorescently labeled DNA oligo probes (Raj and Tyagi, 2010). Using this scheme, mRNA of length shorter than 200 nucleotides cannot be detected. However, in most experiments, the absolute transcript copy number is less informative than the relative copy number. To detect short transcripts or sequences, a short single DNA oligo probe can be used. The detection efficiency of a single probe is greater than 50 percent when using a single fluorophore to count mRNA (Wadsworth et al., 2017).

Materials and Reagents

  1. Pyrex bottle (Corning, PYREXTM, catalog number: 13951L )
  2. Falcon tube 50 ml (VWR, catalog number: 89039-658 )
  3. Falcon tube 15 ml (VWR, catalog number: 89039-666 )
  4. Nitrile gloves (VWR, catalog number: 40101-348 )
  5. Light-duty tissue wipers (VWR, catalog number: 82003-820 )
  6. Lens cleaning tissues (Olympus, catalog number: C-0100 )
  7. Aluminum foil
  8. Pipette tips (VWR, catalog numbers: 89079-466 , 89079-460 , and 89079-472 )
  9. Plastic cuvettes (BrandTech Scientific, catalog number: 759075D )
  10. Culture flask (Corning, PYREXTM, catalog number: 4442-250 )
  11. Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-175-C )
  12. Microcentrifuge tube rack (Thermo Fisher Scientific, catalog number: 5973-0015 )
  13. Petri dish (VWR, catalog number: 25384-088 )
  14. #1.5 18 mm square coverslip (Fisher Scientific, catalog number: 12-518-108B )
  15. Glass slide (Fisher Scientific, catalog number: 12-544-1 )
  16. Saccharomyces cerevisiae strains (collaborators or ATCC)
  17. Low Auto Fluorescence Immersion Oil (Thorlabs, catalog number: MOIL-30 )
  18. Ethanol (VWR, catalog number: BDH1156 )
  19. Methanol ≥ 99% ACS Spectrophotometric grade (Sigma-Aldrich, catalog number: 154903-2L )
  20. RNase free water (Quality Biological, catalog number: 351-068-131 )
  21. Fluorophore labeled DNA oligo probes, HPLC purified (Integrated DNA technologies or Eurofins Scientific)
  22. High Strength 5-min Epoxy (Amazon B001QFGTHG)
  23. Zymolyase-20T at 21,000 units/g (Zymolyase-20 T, Seikagaku Business Corporation)
  24. SD Complete (see Recipes)
    1. Carbon, Nitrogen, and Salts (CNS)
      Dextrose (Sigma-Aldrich, catalog number: G8270-25KG )
      Ammonium sulfate (Sigma-Aldrich, catalog number: A4418-5KG )
      Potassium phosphate monobasic (VWR, catalog number: MK710002 )
      Magnesium sulfate (Sigma-Aldrich, catalog number: M2773-500G )
      Sodium chloride (Fisher Scientific, catalog number: S671-500 )
      Calcium chloride (Sigma-Aldrich, catalog number: C3306-250G )
      Biotin (Sigma-Aldrich, catalog number: B4501-1G )
      Calcium pantothenate (Sigma-Aldrich, catalog number: 21210-25G-F )
    2. Vitamins and trace elements (Vitamix)
      Folic acid (Fisher Scientific, catalog number: BP251910 )
      Inositol (Sigma-Aldrich, catalog number: 57569-25G )
      Niacin (Acros Organics, catalog number: 128291000 )
      P-aminobenzoic acid (Sigma-Aldrich, catalog number: A9878-25G )
      Pyridoxine HCl (Acros Organics, catalog number: 150770500 )
      Riboflavin (Sigma-Aldrich, catalog number: R9504-25G )
      Thiamine HCl (Sigma-Aldrich, catalog number: T4625-25G )
      Boric acid (Sigma-Aldrich, catalog number: B6768-500G )
      Copper sulfate (Sigma-Aldrich, catalog number: C1297-100G )
      Potassium iodide (Avantor Performance Materials, catalog number: JT3168-4 )
      Ferric chloride (Acros Organics, catalog number: 217091000 )
      Manganese sulfate (Sigma-Aldrich, catalog number: M7634-100G )
      Sodium molybdate 2 (Sigma-Aldrich, catalog number: 243655-5G )
      Zinc sulfate (Sigma-Aldrich, catalog number: Z4750-100G )
    3. Complete Supplement Mixture (CSM)
      Adenine (Sigma-Aldrich, catalog number: A9126-25G )
      Arginine (Sigma-Aldrich, catalog number: A5131-100G )
      Aspartic acid (Acros Organics, catalog number: 105041000 )
      Histidine (Sigma-Aldrich, catalog number: H8000-25G )
      Isoleucine (Acros Organics, catalog number: 166170250 )
      Leucine (Sigma-Aldrich, catalog number: L8000-100G )
      Lysine (Sigma-Aldrich, catalog number: L5626-100G )
      Methionine (Sigma-Aldrich, catalog number: M9625-25G )
      Phenylalanine (Acros Organics, catalog number: 130311000 )
      Threonine (Acros Organics, catalog number: 138930250 )
      Tryptophan (Acros Organics, catalog number: 140590250 )
      Tyrosine (Acros Organics, catalog number: 140641000 )
      Uracil (Acros Organics, catalog number: 157300250 )
      Valine (Acros Organics, catalog number: 140811000 )
    4. Bacto-agar (BD, catalog number: 214030 )
  25. Buffer B (see Recipes)
    Sorbitol (Sigma-Aldrich, catalog number: S6021-1KG )
    Potassium phosphate (dibasic) (Sigma-Aldrich, catalog number: P3786-500G )
  26. Spheroplasting Buffer (see Recipes)
    Vanadyl ribonucleoside complex (Fisher Scientific, catalog number: 50-812-650 )
  27. Hybridization Buffer (see Recipes)
    Dextran sulfate (Sigma-Aldrich, catalog number: D8906-10G )
    Escherichia coli tRNA (Sigma-Aldrich, catalog number: R1753-500UN )
    BSA (RNase free) (Fisher Scientific, catalog number: BP671-1 )
    20x SSC (RNase free) (Thermo Fisher Scientific, catalog number: AM9763 )
  28. Wash Buffer (see Recipes)
    Formamide (RNase free) (VWR, catalog number: 97061-392 )
  29. Imaging Buffer (see Recipes)
    6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich, catalog number: 238813-1G )
    Tris base (For making 200 mM, pH 8 Tris- HCl) (Fisher Scientific, catalog number: BP152-500 )
    Protocatechuic acid (PCA) (Sigma-Aldrich, catalog number: 08992-50MG )
    Protocatechuate-3,4-dioxygenase (PCD) (Sigma-Aldrich, catalog number: P8279-25UN )

Equipment

  1. Pipettors (e.g., VWR, catalog number: 75786-304 )
  2. x-y translation mount (Thorlabs, catalog number: ST1XY-S )
  3. Fiberport (Thorlabs, catalog number: PAF-X-11-PC-A )
    Note: This product has been superseded by part number PAF2P-11A .
  4. Fiber optic cable (Thorlabs, catalog number: SM450 )
  5. Single mode fiberoptic cable (Thorlabs, catalog number: P5-460B-PCAPC-1 )
  6. Widefield microscope (e.g., Olympus, model: IX81 )
  7. Spectrophotometer (e.g., Eppendorf, catalog number: 2231000516 )
  8. Centrifuge (e.g., Thermo Fisher Scientific, model: SorvallTM LegendTM XTR , catalog number: 75004521)
  9. Lenses (e.g., Thorlabs, catalog numbers: ACN127-020-A , LB1157-A)
  10. Filters and dichroics (e.g., Semrock, catalog numbers: BLP01-635R-25 , FF650-Di01-25x36 )
  11. Micro-centrifuge (Eppendorf, catalog number: 022620100 )
  12. Incubator (e.g., Thermo Fisher Scientific, catalog number: 50125590 )
  13. Autoclave (e.g., YAMATO SCIENTIFIC, catalog number: SM300 )
  14. 60x or 100x high NA objective (e.g., Olympus, UPlanSApo 100X/1.4 Oil)
  15. EMCCD camera (e.g., Andor Technology, model: iXonEM + )
  16. Laser illumination (e.g., solid state laser: Oxxius, model: LCX-532L-100 ; Coherent, catalog number: 1185055 )
  17. Slide translation stage (e.g., Ludl Electronic Products, model: BioPoint2 X-Y Stage )

Software

  1. Melting temperature calculator (IDT, http://www.idtdna.com/calc/analyzer)
  2. RNA folding calculator (Mfold, http://unafold.rna.albany.edu/?q=mfold)
  3. Sequence specificity check (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi)
  4. Microscope control (Micromanager, https://micro-manager.org/)
  5. Spot counting software (Fish-Quant, https://bitbucket.org/muellerflorian/fish_quant)
  6. Matlab

Procedure

  1. Probe design
    DNA oligo probes are designed by selecting an 18-30 nucleotide region in the target mRNA using four criteria.
    1. The choice of probe length should be determined by the RNA-DNA melting temperature of the sequence, which can be done by using the calculator provided on the Integrated DNA Technologies, Inc (IDT) website.
    2. The probe should have minimal secondary structure.
    3. The target sequence should have minimal secondary structure. The secondary structure can be calculated for the RNA by using the Mfold website (Zuker, 2003). A proper choice has the 5’ end of the probe binding inside a loop rather than a stem in the secondary structure of the mRNA.
    4. The probe should be checked for homology with other sequences in the genome using BLAST (Basic Local Alignment Search Tool) (Johnson et al., 2008).
    Choice of fluorophore is dictated by the illumination scheme. Best results were obtained using Cy3 and Cy5 dyes labeled internally (available through IDT). Other labeling schemes such as end labeling work adequately, but are less photostable. In budding yeast, cellular auto-fluorescence is high in the same region of the emission spectrum as dyes excited by 470 nm or 488 nm light (Atto-488 and Alexa 488) and these dyes are not recommended.
    When labeled internally the fluorophore should be positioned 2 nt away from either end.

  2. Microscope design
    Our microscope is a custom-built microscope configured in highly inclined illumination geometry (HILO). For single fluorophores, epi-fluorescence will not generate an adequate signal, Figure 1. It is necessary to have a variable angle geometry on the illumination (most TIR/HILO microscopes have this.) This can be achieved by using a fiber coupled laser or coupling a free space laser to a fiber using a fiberport and a fiber optic cable and moving the fiber output with an x-y translation mount (see Figure 2). Using the x-y translation, the beam can be adjusted away from the center of the back focal plane of the objective. Once this beam is displaced to the critical angle, total internal reflection geometry is achieved. The angle for inclined illumination is less than the critical angle and should be selected for optimum z-sectioning and intensity in the volume of interest.


    Figure 1. Comparing Epi and HILO illumination. A. An epi-fluorescence microscope has the illumination incident (red line) on the sample through the objective and the entire volume of the sample is illuminated. This leads to poor signal to noise for single fluorophores since the widefield microscope collects out of focus light. B. An inclined illumination geometry has the illumination (red line) displaced radially in the back focal plane of the objective so that the light becomes a thin laminated sheet in the sample volume (Tokunaga et al., 2008). This enables z-sectioning and increases signal to noise significantly.


    Figure 2. Schematic of the illumination path. One or more lasers are coupled into a fiber optic cable using a fiberport. This fiber is fitted onto an X-Y translation mount and collimated by a short lens (focal length chosen by the field of view). The tube lens focuses the illumination on the back focal plane. When the X-Y position is adjusted away from the center of the back focal plane the beam becomes inclined through the sample volume.

  3. Sample preparation
    Day 1
    At the end of the day, inoculate yeast cells into 50 ml of liquid media in a culture flask from cells actively growing on a plate.

    Day 2–Cell fixation and permeabilization
    1. Measure Cell OD using a spectrophotometer at OD600 by placing 1 ml of cell culture in a cuvet.
    2. Once cell OD600 has reached 0.6, decant cells into a 50 ml Falcon tube and pelleted by centrifuging at 671 x g for 5 min and aspirated.
    3. Resuspend the pellet in 10 ml of ice cold (4 °C) methanol for 10 min for fixation.
    4. Cells are pelleted and resuspended in ice cold Buffer B twice and aspirated.
    5. Resuspend the cells in 1 ml of Spheroplasting buffer and transfer to a 1.75 ml microcentrifuge tube and add 2 µl of 5 units/µl of zymolyase and gently pipette to mix.
    6. Incubate the cells for 30 min or until the OD600 of 100 µl of cells added to 900 µl of deionized water shows a reduction of 30% from the initial OD after 1 min, which demonstrates cell lysis.
    7. Pellet the cells and aspirate. Centrifuge at no more than 268 x g since they are fragile once they are spheroplasts.
    8. Wash the cells two more times in ice cold Buffer B and aspirate (wash means spin down to a pellet, aspirate liquid and resuspend.)
    9. Resuspend the cells in 1 ml of 70% ethanol and keep at 4 °C for a minimum of 1 h to overnight.

    Day 2 cont./Day 3–hybridization
    1. Pellet cells at 268 x g and wash twice each with 1 ml Wash Buffer and aspirate.
      Note: Wash buffer should be prepared fresh and formamide should be warmed to room temperature before opening.
    2. Dilute the probes to 1 µM in 10 mM Tris-HCl, pH 8.
    3. Prepare a mixture of hybridization buffer and probes based on the working concentration determined by titration (see Notes).
    4. Resuspend cells to a final volume of 100 µl in the probe-hybridization buffer mixture by gentle pipetting.
    5. Then wrap samples with aluminum foil and place in the incubator at 30 °C overnight.

    Day 3/Day4–Slide Preparation
    1. Prepare Imaging Buffer immediately before use.
    2. Wipe the slides with ethanol; or (optional) clean slides and coverslips in a plasma cleaner for 10 min.
      Note: The slide should be clean of dust and other particles by wiping with ethanol. Any air bubbles will severely impact the performance of the Imaging Buffer.
    3. Mix 2.5 µl of Imaging Buffer with 2.5 µl of cells and place on the coverslip.
    4. Place the coverslip on a slide and seal with epoxy along the edges.
      Note: Slides should be kept in a dark place while not on the microscope. Several slides can be prepared simultaneously. Once sealed, the performance of the imaging buffer will not degrade for several hours.

  4. Data acquisition
    Using a microscope as described above, hardware control and the acquisition parameters can be set in the Micromanager software (Edelstein et al., 2014). Z-stack images can be acquired using the multi-dimensional tool in Micromanager for each channel of interest (e.g., DIC, Cy5, etc.).
    1. Acquire images at full region of interest (512 x 512) on an EMCCD (iXonEM +, Andor) at 100 msec exposure times. The pixel size and z-step are chosen to be less than or equal to the Nyquist sampling limit for the shortest wavelength (see Introduction to Fourier Optics 3rd edition, Goodman.).
    2. The Nyquist limit for a widefield microscope is shown by the following equations where the wavelength λemission used is the peak emission of the fluorophore chosen, the angle θ is the half-aperture angle, and n is the index of refraction of the immersion oil.


      These values allow the maximum amount of spatial information to be recorded. Sampling above this limit is acceptable although the resolution between two objects will not be improved. Undersampling will lead to the inability to distinguish between two close fluorophores because the information in one pixel is not coupled to the information in its neighboring pixels.
    3. Set the laser to output 25 mW at the sample plane. For each chamber, the thickness is approximately 2 µm and an appropriate number of z-slices are acquired. An example of the contrast between a negative control and a low copy number strain is shown in Figure 3.


      Figure 3. Contrast between strains. A. One z-slice of a negative control for yEvenus mRNA (e.g., wildtype) is shown. All intensity is due to auto-fluorescence. The yEvenus probes are 26nt in length and labeled internally (sequence in Wadsworth, et al., 2017). B. One z-slice of a strain expressing a low copy number (< 20) of yEvenus mRNA transcripts per cell is shown. mRNA transcripts are targeted with a single Cy5 labeled DNA oligo probe.

Data analysis

The Matlab Image Processing Toolbox was used to analyze the three-dimensional images. In cases where the researcher is unfamiliar with coding, we recommend FISH-quant for its rigor and user-friendly GUI. For systems with very non- uniform illumination, Corrected Intensity Distributions using Regularized Energy minimization (CIDRE) (Smith et al., 2015) can be used to flatten the images. Many functions in the Image Processing Toolbox can be accelerated by simply converting them to a gpuArray () in Matlab with a compatible graphics card (e.g., Nvidia Geforce 1080). An outline of the algorithm used to locate cells and spots is as follows:

  1. Segmentation
    1. Perform edge detection on the sharpest DIC image using the Sobel filter in Matlab.
    2. Connect detected edges using 1 x 4 and 4 x 1 structural elements.
    3. Perform binary morphological erosion and dilation of the image.
    4. Label the detected regions in the binary image using the bwlabeln () function.

  2. Spot detection
    1. Apply a wide Gaussian filter to the image and subtract the result from the raw data as an approximation of background fluorescence.
    2. Apply a Laplacian of Gaussian (LoG) filter to the result to enhance the spots.
    3. Inside each region detected by segmentation, find pixels that are local maxima in the LoG result and call those spot candidates.
    4. Fit each spot candidate with a Gaussian profile and subtract the mean intensity of the annular region around the spot from each pixel included in the spot to get the spot intensity.
    5. Determine some threshold brightness based on the intensity of single spots detected on the surface of the slide.
    6. Count the spots detected in each cell by using logical indexing of the segmented cells on an 3D array where the detected centers are marked as 1 and all other locations are 0.

Notes

  1. Probe design notes
    We have used single probes of lengths between 18-30 nt with both internal labeling and end labeling. When compared to a standard smFISH probe set (Biosearch Technologies), we found that the probes hybridized between 50-70% of the true transcript count (Wadsworth et al., 2017). This seems to have more dependence on melting temperature than length. Further, we found that the modified Cy5 analog Quasar 670 was much dimmer (it has a smaller molar extinction coefficient) so that Cy5 outperformed substantially. Finally, when using single probes that can be added independently of one another, we found that the true transcript count was achieved with 4-5 probes (Wadsworth et al., 2017).

  2. Microscope
    It is not necessary to have a custom-built microscope. The minimum criteria for a microscope to detect single fluorophores is a ~1.4 NA 100X Objective, an EMCCD, a coherent light source with at least 5 mW of power at the sample plane, and variable angle illumination (TIR/HILO.) Any microscope that can be adjusted from epi-fluorescence to total internal reflection geometry is adequate to accomplish HILO. We do not recommend TIR geometry as the yeast samples are general 2-3 µm thick and this is well outside the range of TIR. TIR will only illuminate probes non-specifically bound to the surface and not in the cytoplasm.
    We do not recommend a confocal microscope because the signal is in the 1-2 photon limit that is beyond the capability of most turnkey confocal setups. Also, we do not recommend sCMOS or CCD cameras because of the 1-2 photon regime. We have tested several in-house and had poor results.
    With the Andor EMCCD camera (EM stands for electron multiplying), we can observe single fluorophores in either a HILO or total internal reflection geometry with nominal gain that is between 50-500 depending on the fluorophore. With our illumination setup, we find no reason to expose for longer than 100 msec although exposures of 1 sec to 2 sec are common in literature.

  3. Sample Preparation: Day 2 notes
    1. Spheroplasting is the most critical step in order to get the probes inside the cell. If the population of cells is poorly converted to spheroplasts, there will be a lot of variability in the FISH spots counted. Spheroplasting can also be observed by DIC imaging. The cells should appear more round and there should be cell wall debris in the sample.
    2. While overnight treatment with ethanol is acceptable, we find the most consistent results when cells are hybridized after 1 h of ethanol treatment.

  4. Day 2cont./Day 3 – hybridization notes
    1. When working with a sample in hybridization buffer, a sample volume that is too small will not produce a large enough pellet after hybridization. This is particularly noteworthy when trying to work with initial cell culture volumes of 5 ml to 10 ml. Also, it should be noted that the pellet should be around 10 µl to 20 µl of the hybridization volume. Larger concentrations of cells cause poor efficiency of probe penetration in any dense clusters of cells.
    2. Create serial dilutions of probes in hybridization buffer from 1 nM to 100 nM or a range where the detected number of FISH spots in the sample plateaus. This concentration is chosen as the working concentration of probes. 65 nM working concentration probes seems to be appropriate for probes of 60 °C RNA-DNA melting temperature in the range of 18-26 nt.
    3. When imaging, if there are many probes diffusing in solution around your cells, then the cells need further washing to remove unbound probes.
    4. Commercial anti-fade reagents such as Prolong Gold may work, but have not been tested in our lab for single fluorophore detection.

  5. Day 3/Day4 – Slide preparation notes
    The imaging buffer will not perform well if not used immediately. When imaging, if probes rapidly photobleach as the slide is explored by moving from one field of view to the next with live viewing, which means your imaging buffer is not working due to expiring or due to tiny bubbles in the chamber (they can be seen by eye).
    We avoid using nail polish although it is common, because it contains solvents that will wick into the sample and cause photobleaching or auto-fluorescence. We find epoxy does not have that effect.

  6. Data acquisition notes
    1. We use a 1.4 NA 100x objective with additional magnification to oversample based on the Nyquist criterion. The camera is set to Frame Transfer mode. The hardware binning is set to 1 x 1. The gain is set to a level that confines the intensity to the dynamic range of the camera on the brightest sample (200 for Cy5).
    2. When trouble shooting poor signal to noise, there are several potential sources of auto-fluorescence; methanol, ethanol, and formamide. We found that lower grade methanol and ethanol are fluorescent in the visible spectrum and use ≤ 99.5 ACS grade reagents. Also, if the auto-fluorescence appears during the hybridization procedure, it is likely because of formamide being contaminated. Formamide is very delicate and will convert to formic acid when mixed with water from condensation if handled poorly. This will lead to inconsistent results.

Recipes

Q: The concentration at the end of reagent.

  1. SD Complete (1 L), pH 5.8
    Carbon, Nitrogen, and Salts (CNS) 26.7 g
    Dextrose 20 g
    Ammonium sulfate 5 g
    Potassium phosphate monobasic 1 g
    Magnesium sulfate 0.5 g
    Sodium chloride 0.1 g
    Calcium chloride 0.1 g
    1. Vitamins and trace elements (Vitamix) 2,840 µl
      Biotin (5 mg/50 ml)
      20 µl
      Calcium pantothenate (1 g/50 ml)
      20 µl
      Folic acid (5 mg/50 ml)
      20 µl
      Inositol (0.5 g/50 ml)
      200 µl
      Niacin (0.1 g/50 ml)
      200 µl
      P-aminobenzoic acid (50 mg/50 ml)
      200 µl
      Pyridoxine HCl (1 g/50 ml)
      20 µl
      Riboflavin (5 mg/50 ml)
      2 ml
      Thiamine HCl (1 g/50 ml)
      20 µl
      Boric acid (1.25 g/50 ml)
      20 µl
      Copper sulfate (0.1 g/50 ml)
      20 µl
      Potassium iodide (0.25 g/50 ml)
      20 µl
      Ferric chloride (0.5 g/50 ml)
      20 µl
      Manganese sulfate (1 g/50 ml)
      20 µl
      Sodium molybdate (0.5 g/50 ml)
      20 µl
      Zinc sulfate (1 g/50 ml)
      20 µl
    2. Complete Supplement Mixture (CSM) 790 mg
      Adenine 10 mg
      Arginine 50 mg
      Aspartic acid 80 mg
      Histidine 20 mg
      Isoleucine 50 mg
      Leucine 100 mg
      Lysine 50 mg
      Methionine 20 mg
      Phenylalanine 50 mg
      Threonine 100 mg
      Tryptophan 50 mg
      Tyrosine 50 mg
      Uracil 20 mg
      Valine 140 mg
    3. Bacto-agar 20 g optional
  2. Buffer B (1 L)
    Sorbitol (218 g)
    Potassium phosphate (dibasic) (17.4 g)
    RNase free water
  3. Spheroplasting Buffer (10.1 ml)
    Buffer B 10 ml
    Vanadyl ribonucleoside complex (200 mM) 100 µl
  4. Hybridization Buffer (10 ml)
    Dextran sulfate 1 g
    Escherichia coli tRNA 10 mg
    Vanadyl ribonucleoside complex (200 mM) 100 µl
    BSA 40 µl (5 mg/ml) (RNase free)
    20x SSC 1 ml (RNase free)
    RNase free water
  5. Wash Buffer (50 ml)
    Formamide 5 ml (RNase free)
    20x SSC 5 ml (RNase free)
    RNase free water
  6. Imaging Buffer (100 µl)
    Trolox (1 mM)
    70 µl
    20x SSC
    10 µl
    Tris-HCl (200 mM, pH 8)
    5 µl
    Protocatechuic acid (PCA) (25 mM)
    10 µl
    Protocatechuate-3,4-dioxygenase (PCD)(200 nM)
    5 µl

Acknowledgments

This protocol has been adapted from Raj et al. (2010). This work was supported by Georgia Institute of Technology startup funds, GAANN Molecular Biophysics and Biotechnology Fellowship, and the National Institutes of Health grant (R01-GM112882). The authors declare no conflicts of interests or competing interests.

References

  1. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using muManager software. J Biol Methods 1(2).
  2. Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S. and Madden, T. L. (2008). NCBI BLAST: a better web interface. Nucleic Acids Res 36(Web Server issue): W5-9.
  3. Raj, A. and Tyagi, S. (2010). Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472: 365-386.
  4. Smith, K., Li, Y., Piccinini, F., Csucs, G., Balazs, C., Bevilacqua, A. and Horvath, P. (2015). CIDRE: an illumination-correction method for optical microscopy. Nat Methods 12(5): 404-406.
  5. Tokunaga, M., Imamoto, N. and Sakata-Sogawa, K. (2008). Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2): 159-161.
  6. Wadsworth, G. M., Parikh, R. Y., Choy, J. S. and Kim, H. D. (2017). mRNA detection in budding yeast with single fluorophores. Nucleic Acids Res 45(15): e141.
  7. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13): 3406-3415.

简介

mRNA表达的定量分析是理解细胞状态的重要部分。 RNA荧光原位杂交(FISH)技术涉及用一组40个互补的荧光标记的DNA寡核苷酸探针靶向RNA转录物。 然而,许多情况下,如转录本短于200 nt,剪接变异,或创建转录本的替代起始位点,这些转录本与一组多重探针无法区分。 为此,我们调整了标准FISH方案,以允许使用具有单个荧光团的单个探针来量化出芽酵母细胞内的转录物的量。 除了允许定量短转录本或转录本的短特征之外,该技术还降低了执行FISH的成本。

【背景】通过单分子荧光原位杂交(smFISH)可以精确定量单细胞转录谱。 该过程通过用多个荧光标记的DNA寡核苷酸探针靶向单个mRNA分子给出了良好的噪声信号(Raj和Tyagi,2010)。 使用该方案,不能检测到长度短于200个核苷酸的mRNA。 然而,在大多数实验中,绝对转录本拷贝数比相对拷贝数少。 为了检测短的转录物或序列,可以使用短的单个DNA寡核苷酸探针。 当使用单个荧光团计数mRNA时,单个探针的检测效率大于50%(Wadsworth等人,2017)。

关键字:RNA FISH, 荧光原位杂交, 酿酒酵母, 芽殖酵母, 转录, 单分子

材料和试剂

  1. Pyrex瓶(Corning,PYREX TM,目录号:13951L)
  2. 猎鹰管50毫升(VWR,目录号:89039-658)
  3. Falcon管15毫升(VWR,目录号:89039-666)
  4. 丁腈手套(VWR,目录号:40101-348)
  5. 轻型组织抹布(VWR,目录号:82003-820)
  6. 镜头清洁纸(奥林巴斯,产品目录号:C-0100)
  7. 铝箔
  8. 移液器吸头(VWR,产品目录号:89079-466,89079-460和89079-472)
  9. 塑料比色杯(BrandTech Scientific,目录号:759075D)
  10. 培养瓶(Corning,PYREX TM,目录号:4442-250)
  11. 微量离心管(Corning,Axygen <\ sup>,目录号:MCT-175-C)
  12. 微量离心管架(Thermo Fisher Scientific,目录号:5973-0015)
  13. 培养皿(VWR,目录号:25384-088)
  14. #1.5 18平方毫米盖玻片(Fisher Scientific,目录号:12-518-108B)
  15. 玻璃载玻片(Fisher Scientific,目录号:12-544-1)
  16. 酿酒酵母菌株(合作者或ATCC)
  17. 低自动荧光浸没油(Thorlabs,目录号:MOIL-30)
  18. 乙醇(VWR,目录号:BDH1156)
  19. 甲醇≥99%ACS分光光度级(Sigma-Aldrich,目录号:154903-2L)
  20. RNase free water(Quality Biological,产品目录号:351-068-131)
  21. 荧光标记的DNA寡核苷酸探针,HPLC纯化(Integrated DNA technologies或Eurofins Scientific)
  22. 高强度5分钟环氧树脂(亚马逊B001QFGTHG)
  23. 21,000单位/克的Zymolyase-20T(生化试剂-20 T,生化学工业株式会社)
  24. SD完成(参见食谱)
    1. 碳,氮和盐(CNS)
      葡萄糖(Sigma-Aldrich,目录号:G8270-25KG)
      硫酸铵(Sigma-Aldrich,目录号:A4418-5KG)
      磷酸二氢钾(VWR,目录号:MK710002)
      硫酸镁(Sigma-Aldrich,目录号:M2773-500G)
      氯化钠(Fisher Scientific,目录号:S671-500)
      氯化钙(Sigma-Aldrich,目录号:C3306-250G)
      生物素(Sigma-Aldrich,目录号:B4501-1G)
      泛酸钙(Sigma-Aldrich,目录号:21210-25G-F)
    2. 维生素和微量元素(Vitamix)
      叶酸(Fisher Scientific,目录号:BP251910)
      肌醇(Sigma-Aldrich,目录号:57569-25G)
      烟酸(Acros Organics,目录号:128291000)
      对氨基苯甲酸(Sigma-Aldrich,目录号:A9878-25G)
      盐酸吡哆醇(Acros Organics,目录号:150770500)
      核黄素(Sigma-Aldrich,目录号:R9504-25G)
      盐酸硫胺素(Sigma-Aldrich,目录号:T4625-25G)
      硼酸(Sigma-Aldrich,目录号:B6768-500G)
      硫酸铜(Sigma-Aldrich,目录号:C1297-100G)
      碘化钾(Avantor Performance Materials,目录号:JT3168-4)
      三氯化铁(Acros Organics,目录号:217091000)
      硫酸锰(Sigma-Aldrich,目录号:M7634-100G)
      钼酸钠2(Sigma-Aldrich,目录号:243655-5G)
      硫酸锌(Sigma-Aldrich,目录号:Z4750-100G)
    3. 完全补充混合物(CSM)
      腺嘌呤(Sigma-Aldrich,目录号:A9126-25G)
      精氨酸(Sigma-Aldrich,目录号:A5131-100G)
      天冬氨酸(Acros Organics,目录号:105041000)
      组氨酸(Sigma-Aldrich,目录号:H8000-25G)
      异亮氨酸(Acros Organics,目录号:166170250)
      亮氨酸(Sigma-Aldrich,目录号:L8000-100G)
      赖氨酸(Sigma-Aldrich,目录号:L5626-100G)
      甲硫氨酸(Sigma-Aldrich,目录号:M9625-25G)
      苯丙氨酸(Acros Organics,目录号:130311000)
      苏氨酸(Acros Organics,目录号:138930250)
      色氨酸(Acros Organics,目录号:140590250)
      酪氨酸(Acros Organics,目录号:140641000)
      尿嘧啶(Acros Organics,目录号:157300250)
      缬氨酸(Acros Organics,目录号:140811000)
    4. 细菌琼脂(BD,目录号:214030)
  25. 缓冲区B(见食谱)
    山梨醇(Sigma-Aldrich,目录号:S6021-1KG)
    磷酸氢钾(二元酸)(Sigma-Aldrich,目录号:P3786-500G)
  26. 原生质球缓冲液(见食谱)
    氧钒核糖核苷复合物(Fisher Scientific,目录号:50-812-650)
  27. 杂交缓冲液(见食谱)
    硫酸葡聚糖(Sigma-Aldrich,目录号:D8906-10G)
    大肠杆菌tRNA(Sigma-Aldrich,目录号:R1753-500UN)
    BSA(无RNase)(Fisher Scientific,目录号:BP671-1)
    20x SSC(无RNase)(Thermo Fisher Scientific,目录号:AM9763)
  28. 洗涤缓冲液(见食谱)
    甲酰胺(无RNase)(VWR,目录号:97061-392)
  29. 成像缓冲区(请参阅食谱)
    6-羟基-2,5,7,8-四甲基苯并二氢吡喃-2-羧酸(Trolox)(Sigma-Aldrich,目录号:238813-1G)
    Tris碱(用于制备200mM,pH8 Tris-HCl)(Fisher Scientific,目录号:BP152-500)
    原儿茶酸(PCA)(Sigma-Aldrich,目录号:08992-50MG)
    原儿茶酸酯-3,4-双加氧酶(PCD)(Sigma-Aldrich,目录号:P8279-25UN)

设备

  1. 移液器( ,VWR,目录号:75786-304)
  2. x-y翻译底座(Thorlabs,目录号:ST1XY-S)
  3. Fiberport(Thorlabs,产品目录号:PAF-X-11-PC-A)
    注意:本产品已被零件编号PAF2P-11A取代。
  4. 光缆(Thorlabs,目录号:SM450)
  5. 单模光缆(Thorlabs,目录号:P5-460B-PCAPC-1)
  6. Widefield显微镜(例如,,奥林巴斯,型号:IX81)
  7. 分光光度计(例如,Eppendorf,目录号:2231000516)
  8. 离心机(例如,Thermo Fisher Scientific,型号:Sorvall TM Legend TM XTR,目录号:75004521)
  9. 镜片(例如,Thorlabs,产品目录号:ACN127-020-A,LB1157-A)
  10. 过滤器和二向色性(,例如,Semrock,产品目录号:BLP01-635R-25,FF650-Di01-25x36)
  11. 微型离心机(Eppendorf,目录号:022620100)
  12. 培养箱(如,Thermo Fisher Scientific,目录号:50125590)
  13. 高压灭菌器(,例如,YAMATO SCIENTIFIC,目录号:SM300)
  14. (例如,Olympus,UPlanSApo 100X / 1.4 Oil)
    60x或100x高NA物镜
  15. EMCCD相机(例如,Andor Technology,型号:iXon +)
  16. 激光照射(例如,固态激光器:Oxxius,型号:LCX-532L-100;相干,目录号:1185055)
  17. 幻灯片翻译阶段(,例如,Ludl Electronic Products,型号:BioPoint2 X-Y Stage)

软件

  1. 熔化温度计算器(IDT, http://www.idtdna.com/calc/analyzer )< br />
  2. RNA折叠计算器(Mfold, http://unafold.rna.albany.edu/?q=mfold
  3. 序列特异性检查(BLAST, https://blast.ncbi.nlm.nih.gov/Blast .cgi
  4. 显微镜控制(Micromanager, https://micro-manager.org
  5. 现货统计软件(Fish-Quant, https://bitbucket.org/muellerflorian/fish_quant ) >
  6. Matlab

程序

  1. 探头设计
    通过使用四个标准选择靶mRNA中的18-30个核苷酸的区域来设计DNA寡聚物探针。
    1. 探针长度的选择应由序列的RNA-DNA熔解温度决定,可使用Integrated DNA Technologies,Inc(IDT)网站上提供的计算器完成。
    2. 探针应该有最小的二级结构。
    3. 目标序列应具有最小的二级结构。可以使用Mfold网站(Zuker,2003)计算RNA的二级结构。合适的选择是将探针的5'末端结合在环内,而不是mRNA的二级结构中的茎。
    4. 应使用BLAST(基本局部比对搜索工具)(Johnson等人,2008)检查探针与基因组中其他序列的同源性。
    荧光团的选择由照明方案决定。使用内部标记的Cy3和Cy5染料(可通过IDT获得)获得最佳结果。其他标签计划,如结束标签工作充分,但较不耐用。在芽殖酵母中,细胞自发荧光在发射光谱的相同区域与470 nm或488 nm光(Atto-488和Alexa 488)激发的染料相同,并且不建议使用这些染料。
    当内部标记时,荧光团应位于距离任一端2 nt处。

  2. 显微镜设计
    我们的显微镜是一种定制的显微镜,配置在高度倾斜的照明几何(HILO)中。对于单荧光团,epi-fluorescence不会产生足够的信号,如图1所示。在照明中必须有一个可变角度的几何结构(大多数TIR / HILO显微镜都有这种结构)。这可以通过使用光纤耦合激光器或使用光纤端口和光缆将自由空间激光器耦合到光纤,并使用xy平移支架移动光纤输出(见图2)。使用x-y平移,可以调整光束远离物镜后焦平面的中心。一旦这个光束被移动到临界角,就可以实现全内反射几何。倾斜照明的角度小于临界角度,应该选择最佳的z切片和感兴趣体积的强度。


    图1.比较Epi和HILO照明A. A.落射荧光显微镜在样品上通过物镜照射入射(红线),并照亮样品的整个体积。这导致单个荧光团的信噪比较差,因为宽视场显微镜收集不到聚焦光。 B.倾斜的照明几何结构具有在物镜的后焦平面径向位移的照明(红线),使得光变成样品体积中的薄层压片(Tokunaga等人,2008年)。这使z分段,并显着增加信号噪音。


    图2.照明路径示意图使用光纤端口将一个或多个激光耦合到光纤电缆中。该光纤安装在X-Y平移底座上,并通过短镜头(视场选择的焦距)进行准直。管透镜将照明聚焦在后焦平面上。当X-Y位置远离后焦平面的中心调整时,光束会在样品体积中倾斜。

  3. 样品准备
    第1天
    在一天结束时,将酵母细胞接种到培养瓶中的50ml液体培养基中,培养瓶中的细胞在平板上活跃生长。

    第2天 - 细胞固定和透化
    1. 使用分光光度计在OD 600下通过将1ml细胞培养物置于小杯中测量细胞OD。
    2. 一旦细胞OD 600达到0.6,将细胞倒入50ml Falcon管中并通过在671xg g下离心5分钟并吸出来沉淀。

    3. 在10毫升冰冷(4℃)甲醇中重悬沉淀10分钟以固定。
    4. 将细胞沉淀并重悬于冰冷的缓冲液B中两次并吸出。
    5. 重悬细胞在1毫升球形缓冲液中,并转移到1.75毫升微量离心管中,加入2微升5单位/微升酵解酶并轻轻移液管混匀。
    6. 将细胞孵育30分钟或直至100μl添加至900μl去离子水的细胞的OD 600在1分钟后从初始OD显示减少30%,这表明细胞溶解。 br />
    7. 将细胞沉淀并吸出。
      离心不超过268 em g,因为一旦它们是原生质球,它们就很脆弱。
    8. 在冰冷的缓冲液B中再次洗涤细胞两次,然后抽吸(洗涤意味着旋转成颗粒,吸取液体并重悬)。
    9. 重悬细胞在1毫升的70%乙醇中,并保持在4°C至少1小时过夜。

    第2天继续/第3天杂交
    1. 将沉淀的细胞置于268×g并用1ml洗涤缓冲液和吸出物洗涤两次。
      注意:清洗缓冲液应该新鲜制备,甲酰胺应在打开前加热至室温。
    2. 在10 mM Tris-HCl(pH 8)中将探针稀释至1μM。
    3. 根据滴定测定的工作浓度制备杂交缓冲液和探针的混合物(见注)。

    4. 在探针 - 杂交缓冲液混合物中通过轻轻移液将细胞重悬至终浓度为100μl。
    5. 然后用铝箔包装样品并放置在30°C的培养箱中过夜。

    第3天/第4天 - 幻灯片制作

    1. 在使用前立即准备成像缓冲液
    2. 用乙醇擦拭载玻片;或(可选)在等离子清洁器中清洁幻灯片和盖玻片10分钟。
      注意:幻灯片应该用乙醇擦拭清洁灰尘和其他颗粒。任何气泡都会严重影响成像缓冲器的性能。
    3. 将2.5μl成像缓冲液与2.5μl细胞混合并置于盖玻片上。
    4. 将盖玻片放在一张幻灯片上,沿边缘用环氧树脂密封。
      注意:幻灯片应保存在黑暗的地方,而不要放在显微镜上。可以同时准备几张幻灯片。一旦密封,成像缓冲液的性能不会降低几个小时。

  4. 数据采集
    使用如上所述的显微镜,可以在Micromanager软件中设置硬件控制和采集参数(Edelstein等人,2014)。对于每个感兴趣的通道(例如,DIC,Cy5,等),可以使用Micromanager中的多维工具来获取Z-stack图像。
    1. 在100毫秒的曝光时间,在EMCCD(iXon <,supor>)上采集全部感兴趣区域(512 x 512)的图像。像素大小和z步长选择为小于或等于最短波长的奈奎斯特采样限制(参见傅立叶光学第3版,古德曼简介)。
    2. 宽视场显微镜的奈奎斯特极限由以下等式表示,其中所使用的波长λem <λ发射 是所选荧光团的峰值发射,角度θ是半孔径角度,而n是浸油的折射率。

      “”src
      这些值允许记录最大量的空间信息。尽管两个物体之间的分辨率不会提高,但超过此限制的采样仍然可以接受。欠采样将导致无法区分两个接近的荧光团,因为一个像素中的信息不与其相邻像素中的信息耦合。
    3. 将激光器设置为在样品平面输出25 mW。对于每个腔室,厚度约为2μm,并且获得适当数量的z-切片。图3显示了阴性对照和低拷贝数应变之间的对比例。


      图3.菌株之间的对比A. A.显示yEvenus mRNA阴性对照的一个z切片(例如,野生型)。所有强度都是由于自动荧光。 yEvenus探针的长度为26nt并在内部标记(在Wadsworth, et。,2017)中的序列)。 B.显示每个细胞表达yEvenus mRNA转录物的低拷贝数(<20)的菌株的一个z切片。用单个Cy5标记的DNA寡核苷酸探针靶向mRNA转录物。

数据分析

Matlab图像处理工具箱用于分析三维图像。在研究人员不熟悉编码的情况下,我们推荐FISH-quant用于其严谨和用户友好的GUI。对于光照非常不均匀的系统,可以使用使用正则化能量最小化(CIDRE)(Smith等人,2015年)的校正强度分布来平坦化图像。图像处理工具箱中的许多功能可以通过在兼容的图形卡(例如,Nvidia Geforce 1080)中将它们转换为Matlab中的gpuArray()来加速。用于定位细胞和斑点的算法大纲如下:

  1. 分割

    1. 使用Sobel滤波器在Matlab中对最锐利的DIC图像执行边缘检测。

    2. 使用1 x 4和4 x 1结构元素连接检测到的边缘
    3. 执行二元形态学侵蚀和图像扩大。
    4. 使用bwlabeln()函数在二值图像中标记检测到的区域。

  2. 点检测
    1. 将一个宽的高斯滤波器应用于图像,并从原始数据中减去结果作为背景荧光的近似值。
    2. 将高斯拉普拉斯(LoG)过滤器应用于结果以增强斑点。
    3. 在通过分割检测到的每个区域内,找到LoG结果中局部最大值的像素并调用这些候选点。
    4. 用高斯轮廓拟合每个候选点,并从点中包含的每个像素中减去点周围环形区域的平均强度,以获得点强度。
    5. 根据幻灯片表面检测到的单个斑点的强度确定一些阈值亮度。
    6. 通过在3D阵列上使用分段单元的逻辑索引来计算在每个单元中检测到的斑点,其中检测到的中心被标记为1并且所有其他位置都是0。

笔记

  1. 探针设计说明
    我们使用了长度在18-30 nt之间的单个探针,内部标记和末端标记。与标准smFISH探针组(Biosearch Technologies)相比,我们发现探针与真实转录数量的50-70%杂交(Wadsworth et al。,2017)。这似乎比熔融温度更依赖于长度。此外,我们发现修饰的Cy5类似物Quasar 670非常暗淡(它具有较小的摩尔消光系数),因此Cy5显着优于其。最后,当使用可以彼此独立添加的单个探针时,我们发现用4-5个探针实现了真实的转录物计数(Wadsworth等人,2017)。

  2. 显微镜
    没有必要有一个定制的显微镜。显微镜检测单个荧光团的最低标准是〜1.4 NA 100X物镜,EMCCD,在样品平面上至少具有5 mW功率的相干光源以及可变角度照明(TIR / HILO)。任何显微镜,可以从落射荧光调整到全内反射几何形状足以完成HILO。我们不推荐TIR几何形状,因为酵母样品一般2-3μm厚,这远远超出了TIR的范围。 TIR只会照射非特异性结合到表面而不在细胞质中的探针。
    我们不建议使用共聚焦显微镜,因为信号处于1-2光子限制,超出了大多数交钥匙共焦设置的能力。另外,由于1-2光子体系我们不推荐sCMOS或CCD相机。我们已经测试了几个内部并且效果不佳。
    使用Andor EMCCD相机(EM代表电子倍增),我们可以观察到HILO或全内反射几何体中的单个荧光团,其标称增益介于50-500之间,具体取决于荧光团。使用我们的照明设置,我们发现没有理由曝光时间超过100毫秒,尽管曝光1秒到2秒在文献中很常见。

  3. 样品制备:第2天的注意事项
    1. 原生质球是获得细胞内探针最关键的步骤。如果细胞群体转化为原生质球很少,那么计数的FISH斑点会有很多变异。原生质球化也可以通过DIC成像观察到。细胞应该看起来更圆,样品中应该有细胞壁碎片。
    2. 虽然用乙醇过夜处理是可以接受的,但是当乙醇处理1小时后细胞杂交时,我们发现最一致的结果。

  4. Day 2cont./Day 3 - 杂交说明
    1. 在杂交缓冲液中进行样品处理时,样品体积太小不能在杂交后产生足够大的沉淀。当试图使用5ml至10ml的初始细胞培养物体积时,这是特别值得注意的。另外,应该注意的是,沉淀应该在10μl至20μl的杂交体积中。
      。更大浓度的细胞会导致探针在任何密集的细胞群中渗透效率低下
    2. 在杂交缓冲液中创建连续稀释的探针,范围从1 nM到100 nM,或在样本平台中检测到FISH斑点数量的范围。选择该浓度作为探针的工作浓度。 65 nM工作浓度探针似乎适用于温度范围为18-26 nt的60°C RNA-DNA探针。
    3. 成像时,如果有许多探针在细胞周围的溶液中扩散,则细胞需要进一步清洗以除去未结合的探针。
    4. 商业防褪色试剂如Prolong Gold可能有效,但尚未在我们的实验室中进行单荧光检测。

  5. 第3天/第4天 - 幻灯片准备笔记 如果不立即使用,成像缓冲区的性能不佳。成像时,如果通过实时观看从一个视场移动到下一个视场探测载玻片,探头会迅速产生光漂白,这意味着您的成像缓冲区由于过期或由于室内微小的气泡而不工作(可以看到通过眼睛)。
    我们避免使用指甲油,尽管它很常见,因为它含有溶剂,会吸入样品中引起光漂白或自动荧光。我们发现环氧树脂没有这种效果。

  6. 数据采集笔记
    1. 基于奈奎斯特准则,我们使用1.4 NA 100x物镜,并使用额外的放大倍数进行过采样。相机设置为帧传输模式。硬件分档设置为1 x 1.增益设置为将亮度限制在最亮样本上的相机动态范围(Cy5为200)的水平。
    2. 当遇到信噪比较差的问题时,有几种潜在的自动荧光来源;甲醇,乙醇和甲酰胺。我们发现较低等级的甲醇和乙醇在可见光谱中发荧光,并使用≤99.5 ACS级试剂。另外,如果在杂交过程中出现自发荧光,可能是因为甲酰胺被污染。甲酰胺非常脆弱,如果处理不好,会与冷凝水混合时转化为甲酸。这将导致不一致的结果。

食谱

问:试剂末端的浓度。

  1. SD完全(1 L),pH 5.8
    碳,氮和盐(CNS)26.7克
    葡萄糖20克
    硫酸铵5克
    磷酸二氢钾1克
    硫酸镁0.5克
    氯化钠0.1克
    氯化钙0.1克
    1. 维生素和微量元素(Vitamix)2,840μl
      生物素(5毫克/ 50毫升)
      20微升
      泛酸钙(1克/ 50毫升)
      20微升
      叶酸(5毫克/ 50毫升)
      20微升
      肌醇(0.5克/ 50毫升)
      200微升
      烟酸(0.1克/ 50毫升)
      200微升
      对氨基苯甲酸(50毫克/ 50毫升)
      200微升
      盐酸吡哆醇(1克/ 50毫升)
      20微升
      核黄素(5毫克/ 50毫升)
      2毫升
      硫胺素HCl(1克/ 50毫升)
      20微升
      硼酸(1.25g / 50ml)
      20微升
      硫酸铜(0.1克/ 50毫升)
      20微升
      碘化钾(0.25克/ 50毫升)
      20微升
      三氯化铁(0.5克/ 50毫升)
      20微升
      硫酸锰(1克/ 50毫升)
      20微升
      钼酸钠(0.5克/ 50毫升)
      20微升
      硫酸锌(1克/ 50毫升)
      20微升
    2. 完全补充混合物(CSM)790毫克
      腺嘌呤10毫克
      精氨酸50毫克
      天门冬氨酸80毫克
      组氨酸20毫克
      异亮氨酸50毫克
      亮氨酸100毫克
      赖氨酸50毫克
      蛋氨酸20毫克
      苯丙氨酸50毫克
      苏氨酸100毫克
      色氨酸50毫克
      酪氨酸50毫克
      尿嘧啶20毫克
      缬氨酸140毫克
    3. 细菌琼脂20克可选
    4. 缓冲液B(1 L)
      山梨糖醇(218克)
      磷酸钾(二元酸)(17.4克)
      无RNase的水
    5. 原生质球缓冲液(10.1 ml)
      缓冲液B 10毫升
      氧钒核糖核苷复合物(200 mM)100μl
    6. 杂交缓冲液(10毫升)
      葡聚糖硫酸盐1克
      大肠杆菌tRNA 10 mg
      氧钒核糖核苷复合物(200 mM)100μl
      BSA 40μl(5 mg / ml)(不含RNase)
      20x SSC 1毫升(无RNase)
      无RNase的水
    7. 洗涤缓冲液(50毫升)
      甲酰胺5毫升(无RNase)
      20x SSC 5 ml(不含RNase)
      无RNase的水
    8. 成像缓冲液(100μl)
      Trolox(1 mM)
      70微升
      20x SSC
      10微升
      Tris-HCl(200mM,pH8)
      5微升
      原儿茶酸(PCA)(25 mM)
      10微升
      原儿茶酸酯-3,4-双加氧酶(PCD)(200 nM)
      5微升

      致谢

      该协议已被改编自Raj等人(2010)。佐治亚理工学院启动资金,GAANN分子生物物理学和生物技术奖学金以及国家卫生研究院拨款(R01-GM112882)支持这项工作。作者声明不存在利益冲突或利益冲突。

      参考

      1. Edelstein,A.D.,Tsuchida,M.A.,Amodaj,N.,Pinkard,H.,Vale,R.D。和Stuurman,N.(2014)。 使用muManager软件进行显微镜控制的先进方法 J Biol方法 1(2)。
      2. Johnson,M.,Zaretskaya,I.,Raytselis,Y.,Merezhuk,Y.,McGinnis,S.and Madden,T.L。(2008)。 NCBI BLAST:更好的网络界面 Nucleic Acids Res 36(Web服务器问题):W5-9。
      3. Raj,A。和Tyagi,S。(2010)。 使用多个单独标记的探针原位检测个体内源性RNA转录本。 方法Enzymol 472:365-386。
      4. Smith,K.,Li,Y.,Piccinini,F.,Csucs,G.,Balazs,C.,Bevilacqua,A。和Horvath,P。(2015)。 CIDRE:光学显微镜的照明校正方法 Nat Methods < / 12(5):404-406。
      5. Tokunaga,M.,Imamoto,N.和Sakata-Sogawa,K。(2008)。 高度倾斜的薄照明可在细胞中实现清晰的单分子成像。 Nat方法 5(2):159-161。
      6. Wadsworth,G.M.,Parikh,R.Y.,Choy,J.S。和Kim,H.D。(2017)。 用单荧光基团检测出芽酵母中的mRNA Nucleic Acid Res 45(15):e141。
      7. Zuker,M.(2003)。 用于核酸折叠和杂交预测的Mfold web服务器 Nucleic Acids Res 31(13):3406-3415。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wadsworth, G. M., Parikh, R. Y. and Kim, H. D. (2018). Single-probe RNA FISH in Yeast. Bio-protocol 8(11): e2868. DOI: 10.21769/BioProtoc.2868.
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