TITLE: Modified Arabidopsis Root smRNA FISH Protocol
AUTHORS: Susan Duncan, Hans Johansson

[DESCRIPTION]
Single molecule RNA FISH (smRNA FISH) is an imaging method that labels individual mRNA molecules in cells to facilitate localization and quantitative studies. Here we present a modified protocol for mRNA labelling in Arabidopsis root meristem cells that retains GFP fluorescence. Although the processing steps impact GFP intensity, this protocol demonstrates visualization of RNA together with highly expressed protein. This protocol is presented together with simplified image analysis steps that aim to support inexperienced cell biologists through from experimental set up to mRNA quantification.

[BEFORE_START]
Before starting the fixation step, clean bench surfaces and equipment with RNase Away
Ensure your 5 nmol dried oligonucleotide probe blend has been suspended in 400 mL of TE buffer (10 mM Tris-HCl, 1mM EDTA pH 8.0).  This creates a probe stock of 12.5 micromolar (µM). To minimise freeze thaw cycles  make small aliquots and store them at -20 °C 
Prepare 50 mL 1xPBS using nuclease free 10x PBS and nuclease free water.
 



[GUIDELINES]
When performing Stellaris RNA FISH, it is imperative to limit RNA degradation. You should try to ensure that all consumables and reagents are RNase-free.

[STEPS]
SECTION: Plant Growth
1. Sterilize then sow a row of Col-0 Arabidopsis seeds onto half strength Murashige and Skoog Medium (1/2 MS) near the top of a 10 cm square petri plate.

SECTION: Plant Growth
2. Stratify the seeds at 4 °Cfor two days. 

SECTION: Plant Growth
3. Take the plate out of the cold and place it vertically in a growth cabinet set for 16 hours light and 8 hours dark at a constant 20 °C  (a plate holder can be useful to ensure stability).  

SECTION: Root Fixation
5. Prepare 4 mL  4% fixative in a 3 cm petri dish by diluting 1 mL of 16% methanol free formaldehyde in  3 mL of 1x PBS. 
 


SECTION: Root Fixation
6. Use forceps to remove whole seedlings from the plate and submerge them in the 4% formaldehyde then leave to fix for 30 min.

 

SECTION: Root Fixation
7. Move seedlings into a fresh petri dish and carry out three washes with 1 x PBS. 
SECTION: Sample Preparation
8. Arrange 3 plants on a slide, place a  No 1 coverslip on top. Then, starting at the root tip, squash each seedling  flat using a pencil eraser. 
SECTION: Sample Preparation
9. Whilst holding the seedlings under the coverslip with forceps, carefully immerse in liquid nitrogen for 10 s. 

SECTION: Sample Preparation
10. Immediately after the slide is removed from the liquid nitrogen, flip off the coverslip using a razor blade and carefully scape away the frozen leaves. Leave the slide to air dry for 60 min at Room temperature. 

SECTION: Sample Preparation
11. Immerse the slide in 70% EtOH in a coplin jar for 60 min.

SECTION: Probe Hybridization
12. Remove the slide from the ethanol, tip off residual liquid and leave for 5 min at Room temperature.

SECTION: Probe Hybridization
13. Carry out 1 x 5 min 200 µL wash on the slide with freshly prepared 10 % (v/v) formamide Wash Buffer A.
 


SECTION: Probe Hybridization
14. Thoroughly mix 100 µL  of freshly prepared 10 % (v/v)  formamide Hybridization Buffer with 1 µL of each probe type of interest (dissolved to250 nanomolar (nM) concentration in TE buffer).
 


 


SECTION: Probe Hybridization
15. Add to the sample and cover with a Hybrislip , or other suitable plastic cut to size (good results have been achieved using the inside surface of an unused autoclave bag). 

SECTION: Sample Mounting
17. Wash slide with 200 µL  Wash Buffer A, then add another 200 µL Wash Buffer A and cover with a fresh Hybrislip (or plastic alternative). Return to 37 °Cincubator for 30 mins.

SECTION: Sample Mounting
18. Tip off Wash Buffer A and add 100 µL of 1 1 µg /mL DAPI Solution (diluted in Wash Buffer A) to each slide. Cover again, place back in the hybridization chamber and return to 37 °C incubator for 30 mins.

SECTION: Sample Mounting
19. Tip the DAPI Solution from the slide, then carry out a 200 µL Wash Buffer B wash for a minimum of 5 mins. 

SECTION: Sample Mounting
20. Tip Wash Buffer B off the slide, then add 20 µL of Vectashield mounting media directly onto the root tip area. Carefully cover with a No.1 coverslip and press down firmly with forceps.

SECTION: Imaging
21. A wide-field fluorescence microscope is required to image single molecules of mRNA. A high numerical aperture (>1.3) and ideally 100x oil-immersion objectives are ideal. Strong light sources, such as a mercury or metal-halide lamp are ideal, but successful imaging has also been achieved with LEDs. Filter sets must be appropriate for the fluorophores. An EM-CCD camera is ideal, but images can be also be obtained using a standard cooled CCD camera that is optimized for low-light level imaging, rather than speed. Z stacks should be set up to fully encompass cell depth and 200 nm steps should be used to provide adequate resolution for analysis.

 
 
SECTION: Basic Image Analysis Steps
23. Per cell mRNA counts can be achieved by basic FIJI commands
 
First carry out a Maximum Z projection of the stack: Image -> Stacks -> Z project (include all slices and select maximum intensity from the drop down menu).

For automated image analysis it is essential that acquisition is completed carefully so that stacks do not include images above or below the cells. This is because if full stack projection is completed blindly it can allow high intensity out-of-focus light to impair smFISH spot detection.  If max projection is being completed manually, it is worth specifying the exact top and bottom slices to ensure optimal downstream analysis.

  
SECTION: Plant Growth
4. Leave seeds to germinate and grow for 5 days

SECTION: Basic Image Analysis Steps
27. We apply the Subtract Background command with a 3 pixel rolling ball radius. This radius setting will depend on you camera set up.  We acquire images with 100 x 100 nm pixels so a 3 pixel radius is required to highlight the ~300 nm diameter smFISH spots.

  

SECTION: Basic Image Analysis Steps
28. Highlight the cell again again by selecting it in the ROI manager. 

 
    

SECTION: Basic Image Analysis Steps
30. Cell volumes can also be estimated for mRNA concentration measurements (mRNA per μm3):
Take the area measurement calculated in step 25 and multiply this by the Z depth
(i.e. the number of Z steps used during image acquisition multiplied by 0.2 μm).

SECTION: Probe Hybridization
16. Place the slide in a humid, hybridization chamber to the 37 °C  incubator and leave overnight.
Note: You can make your own hybridization chamber by covering a 10 cm tissue culture plate with black insulation tape. Then line the base evenly with a flat water-saturated paper towels and apply a single layer of Parafilm on top to provide a dry flat surface for the slides. For more details see: 
 

SECTION: Basic Image Analysis Steps
26. Split the DAPI and mRNA probe channels:  Image -> Colour -> Split Channels. 
Then select the probe channel to work on next.

  

SECTION: Basic Image Analysis Steps
24. Use the freehand selection tool to draw around the perimeter of the cell. Select this as a region of interest (ROI) by pressing t. This will add the area to the ROI manager.
 
     

SECTION: Basic Image Analysis Steps
25. Select the ROI on the manager window and click on measure to open up a results window showing the area (μm2). The mean, min and max fluorescence intensity values of this region are also provided for the channel selected.

Note: Be sure to check that the image file has provided FIJI with calibrated data before trusting these values!

    
  

SECTION: Basic Image Analysis Steps
29. Use the Find Maxima command with a 1700 prominence setting (with preview selected) to highlight and quantify all mRNA signals in this cell. This setting should be considered as a starting point and will differ depending on the microscope set up and probe set. To determine the appropriate setting, compare no probe control and probe images (these must have been acquired using the same microscope settings.) Determine the lowest prominence level that retains spot detection in your probe images, but ignores almost all non-specific spots in the no probe controls. As a guide, for all our reference probe sets, we quantified mRNA labels using 1700 prominence setting and this detected an average of one non-specific signal every 3-4 cells.

      

SECTION: Imaging
22. When imaging suitable transgenic lines it is worth checking whether GFP signal has been retained. Below is an example image showing MON1 mRNA in a cell expressing GFP under a 35S promoter.