TITLE: RNA Slide Preparation Protocol (FFPE) for nanostring DSP - GeoMx - Human Breast samples
AUTHORS: Nicolas Martin

[DESCRIPTION]
This protocol is designed for RNA slide preparation for formalin-fixed tissue. 

[GUIDELINES]
This is the default protocol for slide preparation by Nanostring. 

IMPORTANT: 
Take care to maintain nuclease-free conditions. The most significant risk of contamination comes from GeoMx probes and other oligos. We recommend the use of RNase AWAY (Thermo Fisher 7002) for cleaning of all surfaces and equipment, as
it will limit contamination from oligos, GeoMxprobes and nucleases. After using RNase AWAY, allow area/items to air dry completely, or rinse with DEPC-treated water. See manufacturer's instructions for details.

If sections are too large and/or placed off-center, continue with slide preparation as usual. Just before loading the
slide in the instrument slide tray, scrape off the parts of the tissue exceeding the scan area, ensuring the slide gasket and tip calibration area are tissue-free. Scraping off tissue before slide preparation could generate tissue folds that may result in staining or binding artifacts.


Selecting and Sectioning FFPE Samples

 When preparing, sectioning, and storing FFPE blocks for use in the GeoMx DSP Proteinand RNA assays, take care to preserve sample integrity at all steps. The integrity of FFPE samples is impacted
by many factors including time from excision to fixation, storage conditions,
tissue type, and sample age. Samples with poor integrity are likely to give low
signal, particularly in RNA assays.

GeoMx has been validated for sample blocks up to 3 years old prepared from tissues with a cold ischemic
time of less than 1 hour using 10% NBF or similar fixative. For best results, do not use FFPE blocks more than 10 years old. Assay performance will be influenced by tissue blockage and treatment conditions such as cold/warm ischemic time, fixative, and storage.


Selecting FFPE blocks
 
FFPE blocks should meet the following criteria for the best performance with GeoMx DSP assays.

1. Blocks should be fixed in 10% neutral buffered formalin for 18 to 24 hours at room temperature. This applies to tissues less than 0.5 cm in thickness. Larger tissues have not been tested by NanoString and may require longer fixation times.

 2.    Tissues should be fixed immediately after excision for best results. Fixation within one hour post-excision is acceptable
.
 3. Tissues should be thoroughly dehydrated in ethanol gradients prior to embedding in paraffin.

 4. FFPE blocks should be stored at room temperature and ambient humidity.

 5. For best results, do not use FFPE blocks that are more than 10 years old.


Sectioning FFPE blocks

 The following are general guidelines for sectioning FFPE blocks for optimal GeoMx DSP assay performance. This is not meant to be an all- inclusive guide on sectioning. Please refer to your local pathologist or core facility for training on sectioning.

Due to oxidation at the surface, discard the first few sections cut from the block face.

NanoString recommends SuperFrost™ Plus slides (for manual slide preparation) or Apex BOND® or
BOND Plus slides (for BOND automated slide preparation or manual slide preparation for tissues that exhibit poor adhesion).

FFPE tissue sections should be cut 5 µm thick on a calibrated microtome and mounted on the slide immediately, without scratches or folds.


Tissue sections must be placed in the Scan Area (shown in green in  Figure 1) in the center of the slide and be no
larger than 35.3 mm long by 14.1 mm wide.They should not overlap the slide gasket(shown in blue)or the tip calibration area (shown in red). If mounting multiple sections per slide, ensure that tissues are at least 2–3 mm apart and still fit within the ScanArea.


 


 Any water trapped under the wax or tissue section should be removed by gently touching a folded Kimwipe to the corner of the wax section. The Kimwipe should not contact the tissue.

 Air dry mounted slides overnight prior to use. Store slides in a vertical position such that any remaining water can drain away from the tissue section.

Slides stored in a desiccator (or in a sealed container with a desiccant pouch)at 4°C or room temperature have been shown to yield quality results for up to 3 months. Quality of results is tissue and block-dependent and should be tested
empirically.




 















[STEPS]
58. 
Prepare reagents

Prepare the reagents using the dilution instructions (see Table 1).
Use DEPC- treated water for all dilutions. The actual volume of
reagents used in the protocol will vary – the volumes to prepare in Table 1 are suggestions.

Table 1: Reagent prep for RNA slide preparation

     Reagent      Dilution      Storage           95% EtOH      Prepare 500 mL of 95% ethanol by adding 25 mL of DEPC-treated water to 475  mL of 100% ethanol. NanoString recommends to prepare fresh each week.         RT           1X PBS pH 7.4      Prepare 1 L of 1X PBS by combining 100 mL of 10X PBS and 900 mL of  DEPC-  treated water. Do not reuse.         RT                 Proteinase K      Default
  concentration (1 µg/mL) is made by adding
  10 μL  of 20 mg/mL Proteinase K to 200 mL of 1X PBS made with  DEPC-treated water. See  Table  2 for alternative
  concentrations by tissue type.  Note: Prepare fresh  and do not reuse. Take  care to pipette  accurately. Inaccurate concentration of Proteinase K will affect  assay performance.               n/a           NBF stop buffer      Add  6.06 g tris base and 3.75 g glycine to 5 L DEPC-treated water to yield  0.1 M  tris, 0.1 M glycine. Store at RT up to 1 month.         RT           2X SSC      Prepare 1 L of 2X SSC by combining 100 mL of 20X SSC and 900 mL of  DEPC-treated water.         RT           2X SSC-T (optional)      Prepare 250 mL of 2X SSC-T by combining 25 mL of 20X SSC, 2.5 mL of  10%Tween20, and 222.5 mL of DEPC-treated water.         RT           4X SSC      Prepare 1 L of 4X SSC by combining 200 mL of 20X SSC and 800 mL of  DEPC-treated water.         RT        1X Tris-EDTA pH 9.0  (antigen retrieval solution)      Prepare 1 L of 1X Tris-EDTA pH 9.0 by combining 100 mL of 10X Tris-EDTA pH  9.0  and 900 mL of  DEPC-treated water.         RT   



 
 
 
 




 
 



 
 





58. Bake slides with mounted sections in a 60°C drying oven for 30 minutes to 3 hours prior to deparaffinization. Stand slides vertically during baking to allow excess paraffin to flow off. Longer baking times may be necessary for some tissues to sufficiently adhere to the slide (e.g., overnight at 37°C followed by 2-4 hr at 60°C); this should be empirically tested. 



SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes),
3. The steamer, staining jars, 1X Tris- EDTA (pH 9.0) (antigen retrieval solution), DEPC- treated water, water bath and Proteinase K solution are preheated here for their use in a later step.

(To prepare cell pellet samples, use a hot plate set to 85°C, rather than the steamer). 

Fill the steamer reservoir up to the fill line with water. Place two staining jars inside, one containing DEPC-treated water and one containing 1X Tris-EDTA (pH 9.0) (Antigen Retrieval Solution). Ensure sufficient reagent volume to cover slides up to the label. Loosely cover each jar with aluminum foil instead of the jar lid to allow for a thermometer reading in a later step. Preheat the steamer to 100°C. More water may need to be added to the steamer during preheating. Check on the steamer every 30 minutes.

The Nesco steamer takes 1 hour to heat the liquid in the jars to a stable maximum temperature near 100°C. The final temperature can be checked by inserting a digital thermometer through the hole in the steamer's lid into the staining jars.

Alternatively, a pressure cooker may be used for antigen retrieval. However, the pressure cooker must be usewithout pressure (i.e. pressure valve open) during the incubation time.   

Prepare the pressure cooker by adding water to the correct level per the manufacturer's instructions, level 6 and preheating to 115°C with the 1X Tris EDTA (pH9.0) and DEPC water. Once the temperature reaches 110°C, turn off the pressure cooker and release the pressure by leaving the valve open.
SECTION: Perform target retrieval (25 minutes)
5. Needed for this step: Steamer, Staining jars, 1X Tris-EDTA (pH 9.0) (antigen retrieval solution), DEPC- treated water (all preheated in the step 2) and 1X PBS.

Plastic staining jars are recommended for this step to avoid the risk of glass breaking. If using a glass jar, be aware of the risk of glass breaking during incubation or upon removal of the jar to room temperature. 
SECTION: Perform target retrieval (25 minutes)
5.1.  Place an instant-read digital thermometer through the vents in the steamer lid without removing thelid and pierce the aluminum foil covering the 1X Tris-EDTA. Ensure the 1X Tris-EDTA has reached ~99°C. Reset the steamer's timer to ensure consistent heating during incubation, and add more water as needed. 

 
 
 
SECTION: Expose RNA target (10-30 minutes)
6. Expose RNA targets (10–30 minutes).
Needed for this step:  preheated water bath, preheated Proteinase K dilution, and 1X PBS. 


SECTION: Expose RNA target (10-30 minutes)
6.1. Incubate slides in Proteinase K solution at 37°C according to the conditions for different tissue types in Table 2. Proteinase K concentration and incubation times may need to be empirically determined. If the tissue type you wish to use is not listed, start with a concentration of 1 µg/mL for 15 minutes.

Proteinase K digestion conditions were determined based on FFPE tissue blocks meeting the constraints outlined in the sample guidance section. Samples were primarily tumor with minimal normal adjacent tissue. Optimization may be required for your sample types. The values listed above are recommended starting points. Using Proteinase K from vendors other than those specified will require optimization of incubation times and concentration.

SECTION: Expose RNA target (10-30 minutes)
6.2. Wash slides in 1X PBS for 5 minutes. During the wash, ensure that the 10% NBF and NBF Stop Buffer needed in the next step are ready. Proceed to the next step immediately.

 
SECTION: Post-fixation (20 minutes)
7. Postfix: Preserve tissue morphology for soft tissues (20 minutes).



 

SECTION: Post-fixation (20 minutes)
7.1. 
Post-fix the tissue by performing these washes (see Figure 3):

 
 
Slides can be stored in the final 1X PBS wash up to 1 hour at room temperature or 6 hours at 4°C.

 
 
SECTION: In situ hybridization (overnight)
8. Needed for this step: hybridization chamber, hybridization oven, Buffer R, RNA Probe Mix, and 2X SSC or DEPC-treated water.

IMPORTANT:
Probe mixes should be handled in an area separate from nCounter work, NGS library prep, or other GeoMx workflows. GeoMx detection reagents can cross-contaminate probe mixes and give misleading or incorrect results. Areas should be cleaned thoroughly with RNase AWAY after probe mix formulation. Alternatively, handle probe mixes in PCR workstations decontaminated with UV light. Change gloves after handling any probe mixes to avoid cross-contamination.

Due to the high sensitivity of this assay, it is recommended that you change pipette filter tips for every step, change gloves frequently, and use fresh wipes to remove excess liquids.

SECTION: In situ hybridization (overnight)
8.1. Prepare reagents: Warm Buffer R and RNA detection probes to room temperature before opening. (Warming RNA detection probes reduces viscosity, improving pipetting accuracy.) Before use, flick to mix, then spin down. Store unused RNA detection probes at 4°C for up to 6 months or re-freeze.
SECTION: In situ hybridization (overnight)
8.2. Clean the hybridization chamber and other equipment with RNase AWAY and allow to dry, or rinse with DEPC- treated water. The hybridization chamber can be a key source of contamination by oligos. Arrange fresh Kimwipes on the bottom of the chamber and wet with 2X SSC or DEPC- treated water. Kimwipes should be thoroughly damp, but the liquid should not pool. If your chamber is light permeable, wrap the lid in aluminum foil to minimize light exposure.
SECTION: In situ hybridization (overnight)
8.3. Make hybridization solution following for NGS assays or Table 4 for nCounter assays.
Confirm that you use probe mix for manual/semi-automated slide preparation (white label, green/white/amber cap) and not probe mix for fully automated slide prep (yellow label, red cap). Write down the part and lot numbers of the probe set you are using. You will need the part number when loading the slides on the instrument. 

Table 4: Hybridization solution for assays with NGS readout(Atlas = WholeTranscriptome Atlas (WTA), n = number of slides. 
 
        Panel
  Configuration         Buffer R         Atlas Probe Mix      First
  Custom/Add-on Probe Mix      Second
  Custom/Add-on Probe Mix      DEPC-  treated
  H2O         Final Volume        Atlas (WTA, CTA,  or CCA)         200 μL x n         25 μL x n         0         0         25 μL x n         250  μL x n        Atlas + 1 custom/add-on  assay*         200 μL x n         25 μL x n         12.5 μL x n         0         12.5 μL x n         250 μL x n        Atlas + 2 custom/add-on  assays*         200 μL x n         25 μL x n         12.5 μL x n         12.5 μL x n         0         250 μL x n        1  custom assay, standalone (no  Atlas)         200 μL x n         0         12.5 μL x n         0         37.5 μL x n         250 μL x n        2  custom assays, standalone (no  Atlas)         200 μL x n         0         12.5 μL x n         12.5 μL x n         25 μL x n         250 μL x n   

SECTION: In situ hybridization (overnight)
8.4. One at a time, remove slides from 1X PBS, wipe away excess liquid, and set in a hybridization chamber in a horizontal position. Take care not to let the slides dry out.

SECTION: In situ hybridization (overnight)
8.5. Ensure that the Kimwipes and liquid do not contact the slides. Hybridization solution can wick off of the slides if it comes into contact with Kimwipes or liquid. 

SECTION: In situ hybridization (overnight)
8.6.  Add 200 μL hybridization solution to each slide. Take care not to introduce any bubbles.

To avoid bubbles, leave a small residual volume in the pipette tip. If a bubble forms, aspirate it gently with the pipette. Do not touch the tissue with the tip. It is preferable to lose some hybridization solution and remove bubbles than to have bubbles in the solution as long as sufficient solution remains to cover the tissue after the coverslip is applied.

IMPORTANT: From this point on, minimize the slides' exposure to light to preserve the integrity of the photocleavable barcodes.


SECTION: In situ hybridization (overnight)
8.7. Gently apply a Grace Bio-Labs HybriSlip
Start by setting one edge of the coverslipdown in solution on the slide, then gradually laying down the rest of the coverslipto avoid the formation of air bubbles (see Figure 4).

 
SECTION: In situ hybridization (overnight)
8.8. Repeat steps 4–7 for each slide.



SECTION: In situ hybridization (overnight)
9. Close hybridization chamber, insert into hybridization oven, and clamp into place. Incubate at 37°C overnight (16–24 hours).
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
10. Needed for this step: water bath, 4X SSC, 100% formamide, 2X SSC, and (optional) 2X SSC-T

 WARNING: Use of appropriate personal protective equipment is advised.

 
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
10.1. Preheat water bath to 37°C.
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
10.2. Warm 100% formamide to room temperature before opening. Make Stringent Wash by mixing equal parts 4X SSC and 100% formamide. Fill two staining jars with Stringent Wash and preheat them in the 37°C water bath.
 
 
SECTION: Perform stringent washes to remove off-target probes (90 minutes)
10.3. 
Dip slides in 2X SSC allowing coverslips to slide off. Continue to wash steps within 5 minutes.

If coverslips do not come off immediately, move them to 2X SSC-T for a maximum of 5 minutes. If coverslips have not fallen off in 5 minutes, proceed to the first Stringent Wash.

 








SECTION: Perform stringent washes to remove off-target probes (90 minutes)
10.4. 
Perform 2 washes in Stringent Wash at 37°C for 25 minutes each, then 2 washes in 2X
SSC for 2 minutes each (see Figure 5). After the last wash,slides can be stored in 2X SSC for up to 1 hour.

 



SECTION: Add morphology markers (100 minutes)
11. Needed for this step: humidity chamber, Buffer W, SYTO 13 nuclear stain, morphology markers, and 2X SSC. 
 

SECTION: Add morphology markers (100 minutes)
11.1. 
Block with Buffer W: Remove one slide at a time from 2X SSC and tap slide on clean, absorbent surface to remove excess liquid. Place slide in the humidity chamber. Cover tissue with up to 200 µL Buffer W and leave at room temperature for 30 minutes, protected from light.




SECTION: Add morphology markers (100 minutes)
11.2. 
Ensure adequate Buffer W surrounds the edges of the tissues so they don't dry out. Use a pipette tip to gently move the solution so there is a 2–3 mm border around the tissue. A hydrophobic barrier or hydrophobic pen can be used at this step if desired but must be carefully removed with a razor blade before loading on the instrument.



SECTION: Add morphology markers (100 minutes)
11.3. Prepare 220 μL of morphology markersolution per slide (see Table 4).

 
* If using non-NanoString morphology markers, optimal concentration in the morphology marker solution must
be determined by user testing.
**If using a different number of morphology markers, Buffer W amount needs to be adjusted to bring total volume up to 220 µL per slide.


SECTION: Add morphology markers (100 minutes)
11.4. Mix morphology marker solution by flicking and briefly picofuging.

SECTION: Add morphology markers (100 minutes)
11.5. Remove Buffer W from one slide at a time by tapping slide onto a Kimwipe, then return the slide to the humidity chamber.
SECTION: Add morphology markers (100 minutes)
11.6. Cover tissue with morphology marker solution (up to 200 µL). 

 

SECTION: Add morphology markers (100 minutes)
11.7. Repeat steps 11.5 and 11.6 for each slide.
SECTION: Add morphology markers (100 minutes)
11.8. Incubate for 1 hour in the humidity chamber at room temperature, protected
from light.
SECTION: Add morphology markers (100 minutes)
11.9. After staining, remove solution by tapping slide on a clean, absorbent surface.


11.10. Wash in 2X SSC, 2 times for 5 minutes each.

 
SECTION: Stripping and re-probing procedure for RNA slides
15. GeoMx RNA assay slides are reusable and can be restained with a different commercial or custom panel following this procedure. This procedure requires a UV light box or transilluminator capable of emitting 302/312 nm UV light (example), 2X SSC-T, 1X Tris-EDTA, and 2X SSC.

SECTION: Stripping and re-probing procedure for RNA slides
15.1.  Place the slide flat on the surface of a UV transilluminator.


SECTION: Stripping and re-probing procedure for RNA slides
15.2. Apply enough 2X SSC-T to completely cover the tissue (50–200μL depending on the size of the tissue).



SECTION: Stripping and re-probing procedure for RNA slides
15.3. Expose to UV light for 3 minutes to cleave tags from bound probes.
SECTION: Stripping and re-probing procedure for RNA slides
15.4. Carefully tap each slide on a clean, disposable surface (e.g., paper towel) to remove liquid and avoid oligo contamination.
SECTION: Stripping and re-probing procedure for RNA slides
15.5. Wash slides by dipping in a staining jar with 2X SSC-T.
SECTION: Stripping and re-probing procedure for RNA slides
15.6. Transfer to another staining jar with fresh 2X SSC-T.

SECTION: Stripping and re-probing procedure for RNA slides
15.7. Incubate the slides in 1X Tris- EDTA at 85°C using either a hotplate or a pressure cooker on a low
pressure setting for 15 minutes.

SECTION: Stripping and re-probing procedure for RNA slides
15.8. Wash 3 times in 2X SSC.
SECTION: Stripping and re-probing procedure for RNA slides
15.9. Proceed to In situ hybridization (overnight) section 8.


SECTION: Load slides onto the GeoMx DSP following the GeoMx DSP Instrument User Manual (MAN-10152).
16. Follow instruction from User Manual 
SECTION: Slides sectioning
1. Tissue sections should be 5 µm thick and mounted on Superfrost Plus or BOND Plus slides. Tissue sections must be placed in the Scan Area (shown in green in Figure 3) in the center of the slide and be no larger than 35.3 mm long by 14.1 mm wide. Mounted material should not overlap the slide gasket (shown in blue) or the tip calibration area (shown in red). If mounting multiple sections per slide, ensure that tissues are at least 2–3 mm apart and still fit within the Scan Area.
 
If sections are too large and/or placed off-center, continue with slide preparation as usual. Just before loading the slide in the instrument slide tray, scrape off the parts of the tissue exceeding the scan area, ensuring the slide gasket and tip calibration area are tissue-free. Scraping off tissue before slide preparation could generate tissue folds that may result in staining or binding artifacts.

 
SECTION: Slides baking
2. Bake slides with mounted sections in a 60°C drying oven for 30 minutes to 3 hours prior to deparaffinization. Stand slides vertically during baking to allow excess paraffin to flow off. Longer baking times may be necessary for some tissues to sufficiently adhere to the slide (e.g., overnight
at 37°C followed by 2-4 hr at 60°C); this should be empirically tested.

SECTION: Perform target retrieval (25 minutes)
5.2. Once the 1X Tris-EDTA reaches 99°C, carefully remove the steamer lid, pressure cooker lid, and jar covers. Dip the slides into the DEPC-treated water for 10 seconds to bring the slide temperature up to ~99°C. Quickly transfer the slides to the 1X Tris-EDTA. Replace jar cover, then replace steamer/ pressure cooker lid.

If using a pressure cooker, ensure that the pressure valve is open and the temperature of the pressure cooker is above 110°C. Once the pressure is fully release, open the pressure cooker and Dip the slides into the DEPC-treated water for 10 seconds to bring the slide temperature up to ~99°C. Quickly transfer the slides to the 1X Tris-EDTA. Replace jar cover, then replace steamer/ pressure cooker lid.

 
SECTION: Perform target retrieval (25 minutes)
5.3. Incubate the slides according to the conditions for different tissue types in Table 2. Incubation times and temperatures may need to be empirically determined. If the tissue type you wish to use is not listed, start with 15 minutes in your empirical testing.

Epitope retrieval times were determined based on FFPE tissue blocks meeting the constraints outlined in the sample guidance section. Samples were primarily tumor with minimal normal adjacent tissue. These conditions may vary by sample,the amount of normal adjacent tissue, and other factors. These conditions were optimized for large tumor sections and may not apply to arrayed tissues, cored tissues, and needle biopsies.

SECTION: Perform target retrieval (25 minutes)
5.4. When target retrieval time is up, move slides to room temperature 1X PBS immediately. 
Wash in room temperature 1X PBS for 5 minutes. Slides can be stored forup to 1 hour in 1X PBS

 
SECTION: Deparaffinize and rehydrate FFPE tissue sections (31 minutes),
4. Deparaffinize and rehydrate FFPE tissue sections. Place slides in a rack and perform the  following washes in stainingjars (see Figure 2). Ensureyou have sufficient buffer volume to cover all slides. Slides should be dipped up and down gently several times when placing in and before removing from staining jars. Afterthe last wash,slides can be stored in the 1X PBS for up to one hour.

 
 
During wash steps, preheat the water bath to 37°C. Prepare the Proteinase K dilution, if not yet done, and add the diluted Proteinase K solution to a staining jar and place in the water bath
to preheat to 37°C. Refer to Table 2for the recommended Proteinase K concentration for your tissue type.

SECTION: Add nuclei morphology markers (25minutes)
12. Cover tissue with diluted nuclei morphology marker solution (200-1000 µL of the diluted nuclei marker). 

Syto 13  FITC 1:10,000
Syto 83  Cy3 1:10,000
Syto 59 Texas Red 1:10,000
Syto 62 Cy 5 1:10,000 
 
SECTION: Add nuclei morphology markers (25minutes)
13. Incubate for 10 min in the humidity chamber at room temperature, protected from light.
14. Wash in DEPC water, 3 times for 5 minutes each.