Single Molecule RNA FISH in Arabidopsis Root Cells

Materials and Reagents

1. 1.5 ml microcentrifuge tube
   
2. Sterile Sterilin^TM 10 cm square Petri dishes for plant growth media (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 109 )
   
3. 22 x 22 mm No. 1 glass coverslips (VWR, catalog number: 631-0124 )
   
4. Poly-L-Lysine slides (Sigma-Aldrich, catalog number: P0425 )
   
5. Razor blades (Agar Scientific, catalog number: AGT586 )
   
6. Parafilm M^® sealing film (Bemis, catalog number: PM992 )
   
7. 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
   
   
8. Low stender-form preparation dishes (VWR, catalog number: 470144-866 ; or similar rimmed glass dish)
   
9. Arabidopsis thaliana roots
   
10. Sodium hypochlorite (NaClO) (VWR, BDH^®, catalog number: CABDH7038-4L )
    
11. Sucrose (Sigma-Aldrich, catalog number: S9378-1KG )
    
12. Phytagel (Sigma-Aldrich, catalog number: P8169 )
    
13. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    
14. Nuclease-free phosphate buffered saline solution (PBS, 10x) pH 7.4 (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9624 )
    
15. Liquid nitrogen
    
16. Ethanol suitable for molecular biology
    
17. Nuclease-free 20x saline-sodium citrate (20x SSC) (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9763 )
    
18. Clear nail varnish (Electron Microscopy Sciences, catalog number: 72180 ; or similar)
    
19. Dextran sulphate (Sigma-Aldrich, catalog number: RES2029D )
    
20. 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.
    
21. Murashige & Skoog basal medium with vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories^®, catalog number: M519 )
    
22. Nuclease-free Tris-EDTA buffer solution (10 mM Tris-HCl, 1 mM EDTA pH 8) (Sigma-Aldrich, catalog number: 93283 )
    
23. DAPI (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D1306 )
    
24. Glucose (Sigma-Aldrich, catalog number: 158968-500G )
    
25. Tris HCl buffer 1 M pH 8, nuclease-free (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9855G )
    
26. Glucose oxidase (Sigma-Aldrich, catalog number: G0543 )
    
27. Bovine liver catalase (Sigma-Aldrich, catalog number: C3155 )
    
28. Deionized formamide (Sigma-Aldrich, catalog number: F9037 )
    
29. 1 N HCl (Sigma-Aldrich, catalog number: 71763 )
    
30. 10% (v/v) bleach (Vortex 5-10% sodium hypochlorite, Procter & Gamble, UK) diluted in dH₂O
    
31. TE buffer (10 nM Tris-HCl, 1 mM EDTA, pH 8.0)
    
32. Nuclease-free water - not DEPC treated (QIAGEN, catalog number: 129117 )
    
33. Murashige and Skoog medium (see Recipes)
    
34. Wash buffer (see Recipes)
    
35. Hybridization solution (see Recipes)
    
36. DAPI solution (see Recipes)
    
37. Anti-fade GLOX buffer (minus enzymes) (see Recipes)
    
38. Anti-fade GLOX buffer (containing enzymes) (see Recipes)
    

Equipment

1. Forceps (Fisher Scientific, S Murray^TM, catalog number: E017/01 ; or similar)
   
2. Coplin jar (Sigma-Aldrich, catalog number: S6016 ; or similar)
   
3. Plant growth chamber (Panasonic Healthcare, Sanyo, catalog number: MLR-352H-PE ; or similar)
   
4. Fume cupboard (Labconco, catalog number: 2247300 ; or similar)
   
5. Laminar flow cabinet (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraguard^TM ECO Clean Bench Floor Stands , catalog number: 50109313; or similar)
   
6. Laboratory oven (Cole-Palmer, catalog number: WZ-52412-83 ; or similar)
   
7. Orbital shaker (Cole-Palmer, catalog number: WZ-51700-13 ; or similar)
   
8. 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.
   
9. A high numerical aperture (> 1.3) and 60 or 100x oil-immersion objective
   
10. Strong light source, such as a mercury or metal-halide lamp (Xenon or LEDs are typically not bright enough)
    
11. Filter sets appropriate for the fluorophores
    

Software

1. ImageJ software: http://rsbweb.nih.gov/ij/index.html (Schindelin et al., 2015)
   
2. FISHcount: https://github.com/JIC-CSB/FISHcount
   
3. Graphpad Prism Software or MS Excel
   

Procedure