RNAscope® HiPlex assay using an automated slide scanner

Materials 

1. RNAscope® HiPlex 50X Probes (user-specified)
2. RNAscope® HiPlex 12 Positive Control Probe-Hs (ACD Cat. No. 324321)
3. RNAscope® HiPlex 12 Negative Control Probe (ACD Cat. No. 324341)
4. RNAscope® HiPlex Protease IV (ACD Cat. No. 322340)
5. RNAscope® HiPlex8 Detection Kit (ACD Cat. No. 324110)
6. Wash Buffer Reagents (ACD Cat. No. 310091)
7. Cleaving Reagents (ACD Cat. No. 324130)
8. HybEZ™ Humidity Control Tray (with lid) (ACD Cat. No. 310012)
9. ACD EZ-Batch™ Wash Tray (ACD Cat. No. 321717)
10. ACD EZ-Batch™ Slide Holder (ACD Cat. No. 321716)
11. HybEZ™ Humidifying paper (ACD Cat. No. 310015)
12. ProLong Gold Antifade Mountant (Fisher Scientific Cat. No. P36930)
13. 10X PBS (Fisher Scientific Cat. No. BP3991)
14. 10% Tween-20 (Fisher Scientific Cat. No. PI85115)
15. 20X SSC (Fisher Scientific Cat. No. BP1325)
16. Carboy
17. Distilled water
18. Tubes (50ml, 15ml, 1.5ml, various sizes)
19. Paper towel
20. Serological pipets (25ml, 10ml, 5ml)
21. 100% ethanol (molecular Grade)
22. 4% paraformaldehyde (PFA)
23. ImmEdge™ Hydrophobic Barrier Pen
24. Coverslip #1.5 high performance (Carl Zeiss, 474030-9000-000)

Equipment

1. HybEZ™ II Oven (ACD Cat. No. 321720)
2. ZEISS Axio Scan.Z1 slide scanner microscopy (SBMS imaging facility)

Note : Please refer 'UQ_GIH_RNAscope.pdf' at the side for full details of the procedure, including 'critical points' and ' Occupational Health and Safety'

RNAscope HiPlex assay workflow

*, optional stopping point (1): Slides may be stored in 100 % ethanol at -20 °C for up to 1 week. Prolonged storage may degrade sample RNA

**, optional stopping point (2): You can store the slides in 5X SSC overnight at RT. Before continuing with the assay, wash the slides twice with 1X Wash Buffer for 2 min at RT.

Figure 1.; Conventional diagnosis tool for cancer. Histopathological annotation via H&E staining showing cancer regions in human Basal Carcinoma Cancer (BCC) tissue (5 µm tissue thickness).

Figure 2. RNAscope® Hiplex assay using customized probes to characterize disease. Shown here are the five user specified probes for BCC sample to study gene signatures with single cell resolution in a fresh frozen section (10 µm tissue thickness). Axio Scan.Z1 Digital Slide Scanner (Zeiss) with 40X objective was used for imaging.

Figure 3. Detecting single RNA molecule expression of the genes of interest at a cellular level using RNAscope® Hiplex assay, with THY1 in orange, IL34 in red and CSFR1 in pink. Cell nuclei were counterstained with DAPI (Blue).

• Weak/no signal with excellent morphology: This may be due to over-fixed tissue, but under digested with protease, leading poor probe accessibility. 
• Poor tissue morphology with high background or non-uniform strong/weak signal: This may be due to under-fixed, but over digested tissue, leading loss of RNA due to protease over digestion. 
• Failing to register and overlay images using RNAScope HiPlex Registration Software: This may be the cause of the location of the same region are not matched between two image rounds. This can be resolved by using a microscope with a precise stage recorder. You can develop and run template matching algorithm to merge images manually. 

• ACD RNAscope HiPlex Assay user manual  (PDF, 1 MB)
• ACD RNAscope HiPlex Image Registration Software user manual (PDF, 647.1 KB)

We acknowledge Ian Fraser's Group for providing human BCC samples for this project. We also thank Dr Shaun Walter at SBMS Imaging facility for his valuable input and expertise in using a Slide Scanner Axio Scan. Z1.

• SBMS Histology faciilty 
• SBMS Imaging facility