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Sep 2017

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Dual-probe RNA FRET-FISH in Yeast
酵母菌双探针RNA FRET-FISH   

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Abstract

mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure processes such as alternate initiation or splicing variation of the transcript. Unlike in a conventional FISH method using multiple probes to target a single transcript, FRET is limited to the use of two probes labeled with matched dyes and requires the use of sensitized emission. Any widefield microscope capable of sensitive single molecule detection of Cy3 and Cy5 should be able to measure FRET in yeast cells. Alternatively, a FRET-FISH method can be used to unambiguously ascertain identity of the transcript without the use of a guide probe set used in other FISH techniques.

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

Quantification of the transcript distribution of single cells is typically accomplished by targeting mRNA with multiple probes to achieve a bright signal that can be distinguished from non-specifically bound probes (Raj and Tyagi, 2010). However, in some instances, there are features on the transcript such as splicing variants or alternative initiation sites that would be indistinguishable to a conventional FISH probe set. These isoform sequences can have short 50 nt uniquely identifying sequences. Using two probes, one can target either side of the junction with a FRET pair and quantify up to three classifications of mRNA isoform simultaneously, e.g., the isoform with both probes (FRET), the isoform with probe 1 only, and the isoform with probe 2 only. The reliance on a single fluorophore or pair of fluorophores requires sensitive detection through an EMCCD. Also, the detection efficiency of a probe for a sequence without other isoforms can be estimated using a FRET pair (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. Flourophore 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. Centrifuge (e.g., Thermo Fisher Scientific, model: SorvallTM LegendTM XTR , catalog number: 75004521)
  3. Spectrophotometer (e.g., Eppendorf, catalog number: 2231000516 )
  4. Micro-centrifuge (Eppendorf, catalog number: 022620100 )
  5. Incubator (e.g., Thermo Fisher Scientific, catalog number: 50125590 )
  6. Autoclave (e.g., YAMATO SCIENTIFIC, catalog number: SM300 )
  7. TIRF/HILO widefield microscope capable of sensitized emission
    1. Lenses (e.g., Thorlabs, catalog numbers: ACN127-020-A , LB1157-A)
    2. Filters and dichroics (e.g., Semrock, catalog numbers: BLP01-635R-25 , FF650-Di01-25x36 , FF560/659-Di01-25x36 , FF01-593/40-25 )
    3. Adjustable mechanical slit (Thorlabs, catalog number: VA100 )
    4. Broadband mirrors (Thorlabs, catalog number: BB1-E02 )
    5. Optical mounts, posts, post holders (Thorlabs)
    6. Widefield microscope (e.g., Olympus, model: IX81 )
    7. 60x or 100x high NA objective (e.g., Olympus UPlanSApo 100X/1.4 Oil)
    8. EMCCD camera (e.g., Andor Technology, model: iXonEM + )
    9. Fiberport (Thorlabs, catalog number: PAF-X-11-PC-A )
    10. Single mode fiberoptic cable (Thorlabs, catalog number: P5-460B-PCAPC-1 )
    11. Laser illumination (e.g., solid state laser: Oxxius, model: LCX-532L-100 ; Coherent, catalog number: 1185055 )
    12. 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
    Two DNA oligo probe sequences are chosen based on the target of interest. Designing a FRET pair of probes is much more constrained than designing a single probe.
    1. The probes should target a specific feature of the mRNA. Ideally, for mRNA isoforms, they should flank the junction between a sequence that is unique to one isoform and a sequence that is conserved in both isoforms. The donor should be on the conserved sequence so that both isoforms can be detected using a single excitation. For example, an intron retention isoform could be detected by placing Cy3 on the exon and Cy5 on the intron. However, FRET can also be used to simply exclude false positives and not need a specific feature. In this case, FRET only occurs if the donor and acceptor probes are on the target so that any spot appearing in the donor channel is a non-specific spot. (Note A1)
    2. The probes should not interfere with the binding of each other. To avoid steric effects, these probes are placed two nucleotides apart from the 3’ end of the first to 5’ end of the second probe.
    3. The probe should be stable and specific. Overall probe length should be determined by RNA-DNA melting temperature (60 °C and between 20-30 nt). (Note A2)
    4. The secondary structure of the target RNA should be avoided. Ideally, one of the probes should bind completely or partially in a loop in the determined mRNA structure (mFOLD) (Zuker et al., 2003).
    5. FRET efficiency should be high. Cy3 and Cy5 should be placed within 5 nm of one another when bound to the target to ensure high FRET. Placing the fluorophores 2 nucleotides in from the adjacent 3’ and 5’ ends of the sequence places the fluorophores ~2 nm apart if there is a 2 nucleotide gap between the ends, e.g., 6 nucleotide total separation.

  2. Microscope design
    Our microscope is a custom-built microscope configured in highly inclined illumination geometry (HILO). This custom microscope is not necessary for imaging single fluorophore considering any widefield TIR/HILO microscope should be able to be configured for this type of imaging (~1-2 photon limit) with a properly sensitive camera, such as an EMCCD camera. For single fluorophores in a FRET pair, epi-fluorescence will not generate an adequate signal, Figure 1. It is necessary to have a variable angle geometry on the illumination. This is essentially the same geometry as a single probe FISH experiment (Wadsworth et al., 2017) with the added requirement of dual channel simultaneous imaging of the signal from the FRET pair (e.g., Cy3 and Cy5). This can be done by using a dual band dichroic (Semrock), which reflects the 532 nm excitation beam, but transmits the emissions of both Cy3 and Cy5. In the emission pathway, the camera is no longer mounted to the microscope. The emission is relayed from the image plane to a conjugate image plane where the EMCCD is positioned. In between these planes are a pair of dichroic mirrors that split the emissions of Cy3 and Cy5 (Semrock) along with filters matched to those fluorophores (Semrock), see Figure 2. These paths can be mapped onto one another using an affine transformation and alignment tools (e.g., DNA probe designed with half FRET efficiency) that fluoresce in both channels. (Note B)


    Figure 1. Comparing Epi and inclined illumination. A. An epi-fluorescence microscope has the illumination incident 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 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. Emission pathway of a microscope configured for two channel FRET. The emission is transmitted through a dichroic with two transmission bands compatible with the FRET pair. An adjustible mechanical slit is placed at the image plane. A dichroic is placed near the image plane formed by the tube lens to split the two bands of emission. Filters are placed in the path between the first dichroic and the second dichroic, which recombines the emissions in the same direction. The broadband mirrors are adjusted so that the light from each path covers half of the sensor. If aligned properly, there should be no difference in orientation or magnification between the two images.

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

    Day 2–cell fixation and permeabilization
    1. Measure cell OD600 using a spectrophotometer at 600 nm by placing 1 ml of cell culture in a cuvette.
    2. Once cell OD600 has reached 0.6, decant cell solution into a 50 ml Falcon tube and pellet by centrifuging at 671 x g for 5 min and aspirate.
    3. Resuspend the pellet in 10 ml of ice cold (4 °C) methanol for 10 min for fixation.
    4. Pellet cells and resuspend in ice cold Buffer B twice and aspirate.
    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. (Note C1)
    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.
    9. Resuspend the cells in 1 ml of 70% ethanol and keep at 4 °C for a minimum of 1 h to overnight. (Note C2)

    Day 2cont./Day 3–hybridization
    1. Pellet cells at 268 x g and wash twice each with 1 ml Wash Buffer and aspirate. Wash buffer should be prepared fresh and formamide should be warmed to room temperature before opening. (Notes D1 and F2)
    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. (Note D2)
    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 to prevent photobleaching and place in an incubator at 30 °C overnight.

    Day 3/Day4–Slide Preparation
    1. Pellet cells at 268 x g and wash twice each with 1 ml Wash Buffer and aspirated. (Note D3)
    2. Imaging buffer is prepared immediately before use. (Note D4)
    3. 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.
    4. Mix 2.5 µl of Imaging Buffer with 2.5 µl of cells and place on the coverslip.
    5. Place the coverslip on a slide and seal with epoxy. (Note E)
      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 (Note F1), 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. Aquire images at full region of interest (512x512) on an EMCCD (iXonEM +, Andor) at 100 msec exposure times. The pixel size and z-step were 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. Set the laser to output 25 mW at the sample plane. For each chamber, the thickness is approximately 2 µm and acquire an appropriate number of z-slices. An example of the contrast between a negative control and a low copy number strain is shown in Figure 3. (Note F2) An example of bleedthrough can be seen in Figure 4.


      Figure 3. Example images of dual-probe FISH-FRET. A. Three channels of a FRET acquisition are shown on a single cell expressing yEvenus mRNA where every target should have both probes. The left and right images are during excitation using 532 nm light. These images represent a single z-slice of Cy5 emission (left) and Cy3 emission (right). Cy5 emission during direct excitation with 640nm light is shown in the center. This image was taken second so that the spots that are visible in the FRET (left) image but not the Cy5 (center) image are primarily due to photobleaching. The spots visible in the Cy3 (right) image are due to a combination of non-specific binding (lower melting temperature of the probe) and lower detection efficiency (inactive fluorophores). B. The 256x512 image of Cy5 under direct (Red) and FRET (Green) emission is shown. Where fluorophores were detected in both channels, the image is yellow. The direct excitation was measured second. Approximately 80% of fluorophores were detected in both under this excitation condition with the difference due to photobleaching during excitation by FRET. Shown above is an example where transcripts are countable (~40 spots per cell). A more extreme example (~1,000 transcripts expected) of FRET detection is shown in Wadsworth et al., 2017. In this example, the control shows that crosstalk between channels is minimal.


      Figure 4. Controls for dual-probe FISH-FRET. Each image shown is at the same scale and contrast. A. Cells that have only been treated with a Cy3 labeled probe are shown in dual-view where the emission on the top and bottom panels of a slice were acquired simultaneously. Some bleedthrough is expected in FRET experiments. Much of the intensity in the bottom channel during 532 nm excitation is due to cellular auto-fluorescence, however, there are some peaks in intensity due to Cy3 emission in the Cy5 channel. B. Cells with only Cy5 labeled probes are shown. Under 532 nm excitation there is very little fluorescence observed. Under 640 nm excitation, Cy5 is seen in the bottom panel and virtually no emission is observed in the top panel. C. In a TIR setup, Cy3 and Cy5 are observed when labeled on the same DNA oligo bound to the surface with BSA-biotin. These are designed to calibrate the affine transformation that maps the top panel to the bottom panel. They can also be used to calibrate sensitized emission to determine FRET efficiency since these fluorophores are separated by a known number of nucleotides and double stranded DNA is essentially rigid below its persistence length. Variations in spot intensity here are due to non-uniform illumination.

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 1x4 and 4x1 structural elements.
    3. Perform binary morphological erosion and dilation of the image.
    4. Label the detected regions 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 a 3D array where the detected centers are marked as 1 and all other locations are 0.

  3. Colocalization
    This step is unnecessary unless interested in the efficiency of hybridization or some other feature requiring colocalization of spots in any of the channels.
    1. Spots are detected in each image.
    2. An affine transformation is calculated to overlay the image by using data taken with an alignment sample bound to the surface in half-FRET condition (Cy3 and Cy5 linked to the same DNA molecule at an intermediate FRET radius), see Figure 4C.
    3. Cy5 spots under FRET and direct excitation are counted as colocalized if they are within a 2 pixel radius. The detection efficiency for each probe can be estimated as follows:

Notes

  1. Probe design notes
    1. The detection efficiency of each probe will be different due to sequence dependence. It should be anticipated in FRET probe pair design that one probe could be detected more efficiently than the other. The efficiency is estimated by counting the spots in each channel as shown in equations (4) and (5). The assumption in this case is that both sequences show up for all target RNA. If the target has isoforms, the assumption fails because the lack of the acceptor no longer implies that it failed to bind. In this case, we can only determine the efficiency of the donor binding. To determine the efficiency of the acceptor, it is necessary to either include a third probe that should colocalize every time the acceptor is detected.
    2. 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 alignment
    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 (EM stands for electron multiplying), we can observe single fluorophores in either a HILO or total internal reflection geometry with nominal gain 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.
    The dual-view setup described here is not necessary, however, it has several advantages over sequential acquisition of acceptor channel and donor channel. These include reducing the time of exposure and thereby reducing the chance of acceptor photobleaching during acquisition. Also, it reduces bleedthrough by introducing dichroism which separate the emission pathways of the two channels.

  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 hour 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. This can also be done with the ratio of FRET probes in the working concentration. We found that a 1:1 ratio was generally adequate, but optimization of this ratio will likely lead to higher colocalization of Cy3 and Cy5 and reduce false negatives.
    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 stick 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 1x1. 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

  1. SD Complete (1 L)
    1. 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
    2. 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
    3. 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
    4. Bacto-agar 20 g optional (requires pH 5.8)
  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 100 µl (200 mM)
  4. Hybridization Buffer (10 ml)
    Dextran sulfate 1 g
    Escherichia coli tRNA 10 mg
    Vanadyl ribonucleoside complex 100 µl (200 mM)
    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

Parts of this protocol have 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).

Competing interests

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. Raj, A. and Tyagi, S. (2010). Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472: 365-386.
  3. 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.
  4. 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.
  5. 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.
  6. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13): 3406-3415.

简介

mRNA荧光原位杂交(FISH)是一种常用于分析细胞中转录物分布的技术。 当与常见的单分子技术荧光共振能量转移(FRET)相结合时,FISH也可用于分析转录本中附近序列的共表达以测量转录本的替代启动或剪接变异等过程。 与使用多个探针靶向单个转录物的常规FISH方法不同,FRET限于使用用匹配染料标记的两个探针,并且需要使用敏化发射。 任何能够灵敏地检测Cy3和Cy5单分子的宽视场显微镜应该能够测量酵母细胞中的FRET。 或者,可以使用FRET-FISH方法明确确定转录本的身份,而不使用其他FISH技术中使用的引导探针组。

【背景】单细胞转录物分布的定量通常通过用多个探针靶向mRNA来实现,以实现可以与非特异性结合的探针区分开的明亮信号(Raj和Tyagi,2010)。但是,在某些情况下,转录本上有特征,例如剪接变体或替代起始位点,这与常规FISH探针组无法区分。这些同种型序列可以具有短的50nt唯一识别序列。使用两种探针,可以使用FRET对定位结合的任一侧,同时定量多达三种mRNA同种型,例如,具有两种探针的同种型(FRET),具有探针1的同种型仅限于探针2的同种型。依赖于单个荧光团或荧光团对需要通过EMCCD进行灵敏检测。而且,可以使用FRET对(Wadsworth等人,2017)来估计没有其他同种型的序列的探针的检测效率。

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. Flourophore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. 21000单位/克的Zymolyase-20T(生化试剂-20T,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的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. 离心机(例如,Thermo Fisher Scientific,型号:Sorvall TM Legend TM XTR,目录号:75004521)
  3. 分光光度计(例如,Eppendorf,目录号:2231000516)
  4. 微型离心机(Eppendorf,目录号:022620100)
  5. 培养箱(如,Thermo Fisher Scientific,目录号:50125590)
  6. 高压灭菌器(,例如,YAMATO SCIENTIFIC,目录号:SM300)
  7. TIRF / HILO宽场显微镜能够敏化发射
    1. 镜片(例如,Thorlabs,产品目录号:ACN127-020-A,LB1157-A)
    2. 过滤器和二向色性(例如,Semrock,目录号:BLP01-635R-25,FF650-Di01-25x36,FF560 / 659-Di01-25x36,FF01-593 / 40-25)
    3. 可调式机械狭缝(Thorlabs,产品目录号:VA100)
    4. 宽带镜子(Thorlabs,产品目录号:BB1-E02)
    5. 光学坐骑,帖子,邮筒(Thorlabs)
    6. Widefield显微镜(例如,,奥林巴斯,型号:IX81)
    7. (例如,Olympus UPlanSApo 100X / 1.4 Oil)
      60x或100x高NA物镜
    8. EMCCD相机(例如,Andor Technology,型号:iXon +)
    9. Fiberport(Thorlabs,产品目录号:PAF-X-11-PC-A)
    10. 单模光缆(Thorlabs,目录号:P5-460B-PCAPC-1)
    11. 激光照射(例如,固态激光器:Oxxius,型号:LCX-532L-100;相干,目录号:1185055)
    12. 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折叠式计算器(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
    根据感兴趣的目标选择两种DNA寡核苷酸探针序列。设计FRET探针对比设计单个探针更受限制。
    1. 探针应该针对mRNA的特定特征。理想情况下,对于mRNA同种型,它们应该位于一种同种型特有的序列与两种同种型中保守的序列之间的连接处。供体应在保守序列上,以便可以使用单一激发来检测两种异构体。例如,可以通过将Cy3置于外显子上并将Cy5置于内含子上来检测内含子保留异构体。然而,FRET也可以用来排除误报,不需要特定的功能。在这种情况下,只有在供体和受体探针位于目标上时才会发生FRET,以便供体通道中出现的任何斑点都是非特异性斑点。 (注A1)
    2. 探针不应该干扰彼此的结合。为避免空间效应,将这些探针置于距第二探针的第一至第5'末端的3'末端两个核苷酸处。
    3. 探针应该是稳定和具体的。总探针长度应由RNA-DNA熔解温度(60℃和20-30nt)确定。 (注A2)
    4. 应避免目标RNA的二级结构。理想情况下,其中一个探针应完全或部分结合在确定的mRNA结构的一个环中(mFOLD)(Zuker et al。 ,2003)。
    5. FRET效率应该很高。当与目标结合时,Cy3和Cy5应置于彼此5nm以内以确保FRET较高。如果在末端之间存在2个核苷酸间隔,例如6个核苷酸的总分离,从序列的相邻3'和5'末端放置2个核苷酸的荧光团使荧光团分开〜2nm。

  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。考虑到任何宽视野TIR / HILO显微镜应该能够使用适当敏感的相机(例如EMCCD相机)配置用于这种类型的成像(〜1-2光子极限),该定制显微镜对于成像单荧光团不是必需的。对于FRET对中的单个荧光团,落射荧光不会产生足够的信号,如图1所示。在照明中必须具有可变角度的几何形状。这实质上与单个探针FISH实验(Wadsworth等人,2017)的几何结构相同,但需要双通道同时成像来自FRET对的信号(例如 Cy3和Cy5)。这可以通过使用反射532nm激发光束但是传输Cy3和Cy5的发射的双频带二向色性(Semrock)来完成。在发射途径中,相机不再安装在显微镜上。发射从像平面中继到EMCCD所在的共轭像平面。在这些平面之间是一对分色镜,将Cy3和Cy5(Semrock)的发射以及与那些荧光团(Semrock)相匹配的滤光片分开,参见图2.这些路径可以使用仿射变换和对齐相互映射( eg ,设计为半FRET效率的DNA探针)在两个通道中发出荧光。 (注意B)


    图1.将Epi和倾斜照明进行比较A.一个落射荧光显微镜具有通过物镜入射到样品上的光照,并且样品的整个体积都被照亮。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.倾斜的照明几何形状使照明在物镜的后焦平面中径向移动,使得光变成样品体积中的薄层压片(Tokunaga等人,2008)。这使z分段,并显着增加信号噪音。


    图2.配置为双通道FRET的显微镜的发射路径发射通过具有与FRET对兼容的两个透射带的二向色透射。一个可调整的机械狭缝放置在图像平面上。二色性放置在由管透镜形成的像平面附近以分开两个发射带。滤光片放置在第一个二向色和第二个二向色之间的路径中,从而将发射重新组合在相同的方向上。调整宽带反射镜,使每条路径的光线覆盖传感器的一半。如果对齐正确,两张图像的方向或放大倍数不应有差异。

  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SD完整液体培养基中,培养瓶中的细胞在SD完整培养板上生长。

    第2天细胞固定和透化
    1. 使用分光光度计在600nm处通过将1ml细胞培养物置于比色杯中测量细胞OD 600。
    2. 一旦细胞OD 600达到0.6,将倾析出的细胞溶液通过在671gxg离心5分钟并抽吸而进入50ml Falcon管和小球中。

    3. 在10毫升冰冷(4℃)甲醇中重悬沉淀10分钟以固定。
    4. 沉淀细胞并在冰冷的缓冲液B中重悬两次并吸出。
    5. 重悬细胞在1毫升球形缓冲液中,转移到1.75毫升微量离心管中,加入2微升5单位/微升裂解酶并轻轻移液管混匀。 (注C1)
    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,然后吸出。
    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。 (注意C2)

    Day 2cont./Day 3-hybridization
    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。清洗缓冲液应新鲜制备,甲酰胺应在开启前升温至室温。 (注D1和F2)
    2. 在10 mM Tris-HCl(pH 8)中将探针稀释至1μM。
    3. 基于通过滴定确定的工作浓度制备杂交缓冲液和探针的混合物。 (注D2)

    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以防止光漂白,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. 将沉淀的细胞置于268gxg并用1ml洗涤缓冲液洗涤两次并吸出。 (注意D3)
    2. 成像缓冲液在使用前立即制备。 (注D4)
    3. 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。
    4. 将2.5μl成像缓冲液与2.5μl细胞混合并置于盖玻片上。
    5. 将盖玻片放在载玻片上并用环氧树脂密封。 (注意E)
      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(注释F1),硬件控制和采集参数可以在Micromanager软件中设置(Edelstein等人,2014年)。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EMCCD(iXon +,Andor)上以100毫秒的曝光时间在全部感兴趣区域(512x512)上拍摄图像。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,Goodman简介)。



    2. 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的例子。(注释F2)渗透的例子可以在图4中看到。



      图3.双探针FISH-FRET的示例性图像A.在表达yEvenus mRNA的单个细胞上显示三个通道的FRET获取,其中每个目标应该具有两种探针。左侧和右侧图像在使用532nm光激发期间。这些图像代表Cy5发射(左)和Cy3发射(右)的单个z切片。中心显示用640nm光直接激发时的Cy5发射。该图像是第二次拍摄的,因此在FRET(左)图像而不是Cy5(中心)图像中可见的斑点主要是由于光漂白。 Cy3(右)图像中可见的斑点归因于非特异性结合(探针的较低解链温度)和较低检测效率(无活性荧光团)的组合。 B.显示直接(红色)和FRET(绿色)发射下的Cy5的256x512图像。在两个通道中检测到荧光团的地方,图像呈黄色。第二次测量直接激发。在这种激发条件下,大约有80%的荧光团被检测到,其中由于FRET激发期间的光漂白造成的差异。以上显示的是转录本可计数的例子(每个细胞〜40个点)。在Wadsworth等人的2017年中展示了一个更极端的例子(预期约1,000个转录本)。在这个例子中,控制显示通道之间的串扰最小。


      图4.双探针FISH-FRET的控件显示的每幅图像都具有相同的比例和对比度。 A.仅用Cy3标记的探针处理的细胞以双视图显示,其中同时获取切片的顶部和底部图像上的发射。在FRET实验中预计会出现一些渗漏现象。 532nm激发期间底部通道中的大部分强度归因于细胞自体荧光,然而,由于Cy5通道中的Cy3发射,在强度上存在一些峰值。 B.仅显示具有Cy5标记的探针的细胞。在532nm激发下,观察到的荧光很少。在640nm激发下,Cy5在底部面板中可见,并且在顶部面板中几乎观察不到发射。 C.在TIR装置中,当在用BSA-生物素结合到表面上的相同DNA寡核苷酸上标记时,观察到Cy3和Cy5。这些设计用于校准将顶部面板映射到底部面板的仿射变换。它们也可用于校准敏化发射以确定FRET效率,因为这些荧光团被已知数量的核苷酸分开,并且双链DNA在其持续长度以下基本上是刚性的。这里斑点强度的变化是由于光照不均匀。

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。对于具有非均匀照明的系统,使用正则化能量最小化(CIDRE)(Smith等人,2015)的校正强度分布可以用于使图像变平坦。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1x4和4x1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()函数标记检测到的区域。

  2. 现场检测
    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。

  3. 共同定位
    除非对杂交效率或某些其他功能感兴趣,否则这些步骤是不必要的,因为这些功能需要在任何频道中进行点的共定位。

    1. 在每幅图像中检测到斑点
    2. 计算仿射变换以通过使用以在半FRET条件下结合到表面的比对样品获取的数据(在中间FRET半径处与同一DNA分子连接的Cy3和Cy5),参见图4C。 >
    3. 在FRET和直接激发下的Cy5斑点如果位于2个像素半径内,则被计数为共定位。每个探针的检测效率可以估算如下:

笔记

  1. 探头设计笔记
    1. 由于序列依赖性,每个探针的检测效率会不同。在FRET探头对设计中应该可以预料,一个探头可以比另一个更有效地被探测到。如方程(4)和(5)所示,通过计算每个通道中的斑点来估计效率。在这种情况下的假设是两个序列都显示出所有的靶RNA。如果目标具有异构体,则该假设失败,因为缺少接受者不再意味着它没有约束力。在这种情况下,我们只能确定供体结合的效率。为了确定受体的效率,有必要包括每次检测到受体时应共同定位的第三个探针。
    2. 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. 显微镜对准
    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,在样品平面至少具有5mW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(EM代表电子倍增),我们可以在HILO或全内反射几何中观察单个荧光团,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。
    这里描述的双视图设置是不必要的,但是,它与顺序采集受体通道和供体通道相比有几个优点。这包括减少曝光时间,从而减少采集过程中受体光漂白的机会。此外,它通过引入分开两个通道的发射路径的二向色来减少渗色。

  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60°C RNA-DNA熔解温度范围为18-26 nt的探针。这也可以用FRET探针在工作浓度中的比例来完成。我们发现1:1的比例通常是足够的,但这个比例的优化可能会导致Cy3和Cy5更高的共定位并减少假阴性。
    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 - 幻灯片准备注意事项
    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,虽然它很常见,因为它含有溶剂,会粘在样品上,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。硬件分箱设置为1x1。增益设置为将亮度限制在最亮样本上摄像机动态范围的水平(Cy5为200)。
    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

  1. SD完成(1 L)
    1. 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
    2. 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
    3. 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
    4. 细菌琼脂20 g可选(需要pH 5.8)
    5. 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
    6. 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100μl(200 mM)
    7. 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100μl(200 mM)
      BSA 40μl(5 mg / ml)(不含RNase)
      20x SSC 1毫升(无RNase)
      无RNase的水
    8. 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
    9. 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

      本协议的部分内容已根据Raj em et al。(2010)进行了修改。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. 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。
      3. 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。
      4. 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。
      5. 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。
      6. 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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引用:Wadsworth, G. M., Parikh, R. Y. and Kim, H. D. (2018). Dual-probe RNA FRET-FISH in Yeast. Bio-protocol 8(11): e2867. DOI: 10.21769/BioProtoc.2867.
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