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Peer-reviewed

Single-probe RNA FISH in Yeast

酵母菌单探针 RNA FISH

Gable M. Wadsworth Gable M. Wadsworth
RP Rasesh Y. Parikh
HK Harold D. Kim

发布: 2018年06月05日第8卷第11期 DOI: 10.21769/BioProtoc.2868 浏览次数: 7870

评审: Gal HaimovichChenchen LiuAnonymous reviewer(s)

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

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-20T, 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

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

分子生物学 > RNA > RNA 检测

微生物学 > 微生物遗传学 > 基因表达

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