酵母菌双探针RNA FRET-FISH

Gable M. Wadsworth; Rasesh Y. Parikh; Harold D. Kim

doi:10.21769/BioProtoc.2867

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Peer-reviewed

Dual-probe RNA FRET-FISH in Yeast

酵母菌双探针RNA FRET-FISH

Gable M. Wadsworth

RP Rasesh Y. Parikh

HK Harold D. Kim email

发布: 2018年06月05日第8卷第11期 DOI: 10.21769/BioProtoc.2867 浏览次数: 7493

评审: Gal HaimovichManuel D GaheteImre Gáspár

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参见作者原研究论文

The authors used this protocol in:

Cover of Nucleic Acids Research, featuring study using the protocol.

Sep 2017

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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

Pyrex bottle (Corning, PYREX^TM, catalog number: 13951L )
Falcon tube 50 ml (VWR, catalog number: 89039-658 )
Falcon tube 15 ml (VWR, catalog number: 89039-666 )
Nitrile gloves (VWR, catalog number: 40101-348 )
Light-duty tissue wipers (VWR, catalog number: 82003-820 )
Lens cleaning tissues (Olympus, catalog number: C-0100 )
Aluminum foil
Pipette tips (VWR, catalog numbers: 89079-466 , 89079-460 , and 89079-472 )
Plastic cuvettes (BrandTech Scientific, catalog number: 759075D )
Culture flask (Corning, PYREX^TM, catalog number: 4442-250 )
Microcentrifuge tube (Corning, Axygen^®, catalog number: MCT-175-C )
Microcentrifuge tube rack (Thermo Fisher Scientific, catalog number: 5973-0015 )
Petri dish (VWR, catalog number: 25384-088 )
#1.5, 18 mm square coverslip (Fisher Scientific, catalog number: 12-518-108B )
Glass slide (Fisher Scientific, catalog number: 12-544-1 )
Saccharomyces cerevisiae strains (collaborators or ATCC)
Low Auto Fluorescence Immersion Oil (Thorlabs, catalog number: MOIL-30 )
Ethanol (VWR, catalog number: BDH1156 )
Methanol ≥ 99% ACS Spectrophotometric grade (Sigma-Aldrich, catalog number: 154903-2L )
RNase free water (Quality Biological, catalog number: 351-068-131 )
Flourophore labeled DNA oligo probes, HPLC purified (Integrated DNA technologies or Eurofins Scientific)
High Strength 5-min Epoxy (Amazon, B001QFGTHG)
Zymolyase-20T at 21 000 units/g (Zymolyase-20T, Seikagaku Business Corporation)
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 )
Buffer B (see Recipes)
Sorbitol (Sigma-Aldrich, catalog number: S6021-1KG )
Potassium phosphate (dibasic) (Sigma-Aldrich, catalog number: P3786-500G )
Spheroplasting Buffer (see Recipes)
Vanadyl ribonucleoside complex (Fisher Scientific, catalog number: 50-812-650 )
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 )
Wash Buffer (see Recipes)
Formamide (RNase free) (VWR, catalog number: 97061-392 )
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

Pipettors (e.g., VWR, catalog number: 75786-304 )
Centrifuge (e.g., Thermo Fisher Scientific, model: Sorvall^TM Legend^TM XTR , catalog number: 75004521)
Spectrophotometer (e.g., Eppendorf, catalog number: 2231000516 )
Micro-centrifuge (Eppendorf, catalog number: 022620100 )
Incubator (e.g., Thermo Fisher Scientific, catalog number: 50125590 )
Autoclave (e.g., YAMATO SCIENTIFIC, catalog number: SM300 )
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: iXon^EM + )
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 )