Single Molecule RNA FISH in Arabidopsis Root Cells
拟南芥根细胞中的单个RNA分子FISH
发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2240 浏览次数: 15276
评审: Gal HaimovichAnca Flavia SavulescuAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2016
Abstract
Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al., 2008). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.
Keywords: Single RNA molecules (单个RNA分子)
Fluorescent in situ hybridization (荧光原位杂交)
Gene expression (基因表达)
Arabidopsis (拟南芥)
Transcription (转录)
Background
While single molecule FISH has been developed to quantitatively measure mRNAs at the single cell level for cultured cells, tissue sections and whole-mount invertebrate organisms, this method was not optimized for use in single cells in plants. Fluorescence imaging in plants is considerably challenging due to endogenous autofluorescence of plant tissues. Here, we report a method to detect single RNA molecules in plants. We describe the detection and automated counting of single transcripts within cells of fixed Arabidopsis root squashes. This method generates isolated cells and single-cell layers, which together with the use of red and far-red dyes maximizes signal-to-noise ratio limiting background noise.
Materials and Reagents
- 1.5 ml microcentrifuge tube
- Sterile SterilinTM 10 cm square Petri dishes for plant growth media (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 109 )
- 22 x 22 mm No. 1 glass coverslips (VWR, catalog number: 631-0124 )
- Poly-L-Lysine slides (Sigma-Aldrich, catalog number: P0425 )
- Razor blades (Agar Scientific, catalog number: AGT586 )
- Parafilm M® sealing film (Bemis, catalog number: PM992 )
- Hybridization chamber
Note: Although these are available commercially (Corning, catalog number: 2551 ) we used 10 cm square Petri dishes covered externally with a layer of black insulation tape (RS Components, catalog number: 494-405 ). A double layer of tissue (KCWW, Kimberly-Clark, catalog number: 7557 ) was then placed in the base and saturated with sterile water before slides were placed on top of a single layer of Parafilm (see Figure 1).
Figure 1. Illustration of the hybridization chambers used for smFISH experiments - Low stender-form preparation dishes (VWR, catalog number: 470144-866 ; or similar rimmed glass dish)
- Arabidopsis thaliana roots
- Sodium hypochlorite (NaClO) (VWR, BDH®, catalog number: CABDH7038-4L )
- Sucrose (Sigma-Aldrich, catalog number: S9378-1KG )
- Phytagel (Sigma-Aldrich, catalog number: P8169 )
- Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
- Nuclease-free phosphate buffered saline solution (PBS, 10x) pH 7.4 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9624 )
- Liquid nitrogen
- Ethanol suitable for molecular biology
- Nuclease-free 20x saline-sodium citrate (20x SSC) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9763 )
- Clear nail varnish (Electron Microscopy Sciences, catalog number: 72180 ; or similar)
- Dextran sulphate (Sigma-Aldrich, catalog number: RES2029D )
- Custom RNA FISH Stellaris® probe sets
We used the online Stellaris Probe Designer to design our smFISH probe sets: https://www.biosearchtech.com/support/education/stellaris-rna-fish. We used a default masking level 2 to avoid general problematic RNA sequences and selected the maximum number of 48 probes, an oligo length of 20 nt and 2 nt minimum spacing between probes.
We also performed TAIR BLAST searches (https://www.arabidopsis.org/Blast/index.jsp) for each probe sequence and considered the results collectively to ensure target specificity. We found Quasar 570 and Quasar 670 dyes equally suitable for imaging RNA in Arabidopsis root cells, however we were unable to detect RNA labelled with Fluorescein modified probes. - Murashige & Skoog basal medium with vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories®, catalog number: M519 )
- Nuclease-free Tris-EDTA buffer solution (10 mM Tris-HCl, 1 mM EDTA pH 8) (Sigma-Aldrich, catalog number: 93283 )
- DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
- Glucose (Sigma-Aldrich, catalog number: 158968-500G )
- Tris HCl buffer 1 M pH 8, nuclease-free (Thermo Fisher Scientific, AmbionTM, catalog number: AM9855G )
- Glucose oxidase (Sigma-Aldrich, catalog number: G0543 )
- Bovine liver catalase (Sigma-Aldrich, catalog number: C3155 )
- Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
- 1 N HCl (Sigma-Aldrich, catalog number: 71763 )
- 10% (v/v) bleach (Vortex 5-10% sodium hypochlorite, Procter & Gamble, UK) diluted in dH2O
- TE buffer (10 nM Tris-HCl, 1 mM EDTA, pH 8.0)
- Nuclease-free water - not DEPC treated (QIAGEN, catalog number: 129117 )
- Murashige and Skoog medium (see Recipes)
- Wash buffer (see Recipes)
- Hybridization solution (see Recipes)
- DAPI solution (see Recipes)
- Anti-fade GLOX buffer (minus enzymes) (see Recipes)
- Anti-fade GLOX buffer (containing enzymes) (see Recipes)
Equipment
- Forceps (Fisher Scientific, S MurrayTM, catalog number: E017/01 ; or similar)
- Coplin jar (Sigma-Aldrich, catalog number: S6016 ; or similar)
- Plant growth chamber (Panasonic Healthcare, Sanyo, catalog number: MLR-352H-PE ; or similar)
- Fume cupboard (Labconco, catalog number: 2247300 ; or similar)
- Laminar flow cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraguardTM ECO Clean Bench Floor Stands , catalog number: 50109313; or similar)
- Laboratory oven (Cole-Palmer, catalog number: WZ-52412-83 ; or similar)
- Orbital shaker (Cole-Palmer, catalog number: WZ-51700-13 ; or similar)
- Wide-field fluorescence microscope
Note: We used an Andor iXon EM-CCD camera (Andor, model: iXon EMCCD Camera ) fitted to a Zeiss, Elyra PS-1 (Zeiss, model: Elyra PS.1 ), however similar images have also been obtained using a standard CCD camera optimized for low light level imaging. - A high numerical aperture (> 1.3) and 60 or 100x oil-immersion objective
- Strong light source, such as a mercury or metal-halide lamp (Xenon or LEDs are typically not bright enough)
- Filter sets appropriate for the fluorophores
Software
- ImageJ software: http://rsbweb.nih.gov/ij/index.html (Schindelin et al., 2015)
- FISHcount: https://github.com/JIC-CSB/FISHcount
- Graphpad Prism Software or MS Excel
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Duncan, S., Olsson, T. S. G., Hartley, M., Dean, C. and Rosa, S. (2017). Single Molecule RNA FISH in Arabidopsis Root Cells. Bio-protocol 7(8): e2240. DOI: 10.21769/BioProtoc.2240.
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分类
植物科学 > 植物分子生物学 > RNA
分子生物学 > RNA > RNA 检测
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