Dec 12, 2023
NanoString GeoMx DSP TMA-TNP protein assay
- Jinho Lee1,
- Jose Ceja Navarro2,
- Koei Chin1,
- Heidi S Feiler1,
- Christopher Corless2
- 1Knight Cancer Institute, OHSU;
- 2Knight Diagnostics Laboratory, OHSU
Protocol Citation: Jinho Lee, Jose Ceja Navarro, Koei Chin, Heidi S Feiler, Christopher Corless 2023. NanoString GeoMx DSP TMA-TNP protein assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyn25pvx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 02, 2022
Last Modified: December 12, 2023
Protocol Integer ID: 63771
Keywords: NanoString, GeoMx, Digital Spatial Profiler, HTAN, TMA, DSP, cell profiling, cell type
Abstract
This protocol outlines the NanoString GeoMx DSP protein assay that was applied in the Human Tumor Atlas Network (HTAN) Tissue MicroArrary (TMA) -TransNetwork Project (TNP).
The TMA-TNP evaluates various characterization and analytics methodologies on a large array of breast tumor samples representing a broad spectrum of disease state and subtype. A commercially available anonymized breast tumor TMA was purchased and serial sections were distributed. Participating HTAN Centers characterized the FFPE specimens using various imaging platforms and generated a spatially resolved cell type/state census using each center’s method of choice. Data was recorded in a common repository to enable joint analysis.
The protocol that immediately precedes this one for TMA-TNP Phase 3 (when this NanoString GeoMx DSP protein assay protocol was first tested) can be found at: dx.doi.org/10.17504/protocols.io.6qpvr6992vmk/v1. It describes FFPE block serial sectioning, slide processing and TMA sample distribution.
In this protocol, the DSP protein assay was performed with the following human marker panels: Immune cell profiling core, Immune activation status, Immune-Oncology (IO) Drug target, Cell death, Pan-tumor, MAPK and PI3K/AKT panels including OHSU custom panel (Cell cycle and DNA damage). A total of 87 protein targets were evaluated in the TMA samples. Three compartments (Immune, Tumor and Stroma) in each TMA core were analyzed to determine cell-to-cell interactions in the tissues.
Materials
1. Materials
Slide Baking Oven
Antigen retrieval pressure cooker
Humidity chamber
Thermocycler
Centrifuge (up tp 2,000g)
2. Reagents
Xylene
Ethanol
TBS
TBS-T
Citrate buffer pH6.0
10% Neutral Buffered Formalin (NBF)
DEPC-treated water
3. Nanostring reagents
GeoMx Instrument Buffer kit / Collection plate (Item no. 100474)
FFPE slide prep kit (Buffer W / Buffer S) (Item no. 121300312)
Human Immune cell profiling core panel (Item no. 121300101)
Human Immune activation status panel (Item no. 121300103)
Human IO Drug target panel (Item no. 121300102)
Human Cell death panel (Item no. 121300112)
Human MAPK panel (Item no. 121300111)
Human PI3K/AKT panel (Item no. 121300113)
Human Pan-tumor panel (Item no. 121300105)
OHSU SQ-35430 custom protein panel
Solid tumor Morphology kit (Anti-PanCK / Anti-CD45 / SYTO-13) (Item no. 121300301)
Hybridization CodeSets (A-H) (Item no. 121300401)
Master kit / Hybridization buffer (Item no. 100052)
FFPE slide sample preparation
FFPE slide sample preparation
1h 59m
1h 59m
FFPE slide preparation
Bake FFPE slides at 60 °C for 01:00:00 .
1h
Deparaffinize by sequential incubation with Xylene (00:03:00 twice), 100% EtOH (00:01:00 ), 95% EtOH (00:01:00 ) and 70% EtOH (00:01:00 ).
9m
Briefly rinse the slides with diH2O (distilled water) and incubate with 1x Citrate antigen retrieval buffer (Epitope retrieval buffer, 6.0 ) for 00:15:00 at high pressure in a pressure cooker.
15m
Take out the slides from the pressure cooker and let them cool down to room temperature around 00:25:00 .
25m
Wash the slides 5 times with TBS-T for 00:02:00 each.
10m
Antibody panel incubation
Antibody panel incubation
1h 59m
1h 59m
DSP protein panel and visualization marker incubation
Mark off the entire tissue section with a hydrophobic pen to create a reagent boundary.
Block the slides for 01:00:00 at room temperature using the Nanostring blocking buffer (Buffer W)
1h
Freshly prepare each commercial protein panel in 1:25 dilution and each visualization marker (PanCK and CD45) in 1:40 dilution with Buffer W to a volume of 220ul per slide.
For the custom antibody panel, the final concentration at 250ng/ml per antibody is calculated and adjusted to the final volume of 220ul.
Incubate slides with all antibody panels and visualization markers for Overnight at 4 °C .
16h
Post fixation and DNA staining
Post fixation and DNA staining
1h 18m
1h 18m
Post fixation
After antibody incubation, wash the slides 3 times with TBS-T for 00:03:00 each.
9m
Fix the slides with 10% NBF (Neutral buffered Formalin) for 00:30:00 at RT.
30m
Wash 3 times with TBS-T for 00:03:00 each, then stain the samples with SYTO-13, prepared by 1:10 dilution in TBS for 00:15:00 at RT.
18m
Slides are briefly washed 2 times with TBS-T for 00:03:00 each.
6m
Loading samples to DSP
Loading samples to DSP
DSP running preparation
Scrape the hydrophobic barrier off with a scalpel or a straight-edged razor.
Place slides on the GeoMx slide tray and clean the back of the slide with 70% EtOH.
Once the gasket is sealed, place 6 mL of Buffer S on slides.
Load the GeoMx slide tray onto DSP platform.
GeoMx operation
GeoMx operation
6h 30m
6h 30m
Slide scan, ROI (Region of Interest) selection and AOI (Area of Interest) segmentation.
Log onto GeoMx software and start with “New / Continue Run”.
After loading slides with the collection plate information, DSP is ready to scan slides.
30m
A slide scan name is created and panel/visualization marker information is selected as below:
In the Probe Reagent Kit field, in any order select Human Immune Cell Profiling Protein Core, Human Immune Activation Status Protein, Human IO Drug Target Protein, Human Cell Death Protein, Human MAPK Signaling Protein, Human Pan-Tumor Protein, Human PI3K/AKT Signaling Protein, and SQ-35430 OHSU Custom
- Select the FITC/525 nm, Cy5/568 nm and Texas Red/615 nm channels.
- For FITC/525 nm, select SYTO 13 as fluorophore, DNA as biological target and enter 50 as exposure time.
- For Cy5/568 nm, select Alexa 532 as fluorophore, PanCK as biological target and enter 300 as exposure time.
- For Texas Red/615 nm, select Alexa 594 as fluorophore, CD45 as biological target and enter 300 as exposure time.
- Select FITC/525 nm as focus channel
10m
When the scan area for each slide has been adjusted with sensitivity setting, select Scan.
30m
ROI selection and AOI segmentation
After scanning is done, each color channel intensity is adjusted to show visualization markers along with tissue or cell line property.
10m
Each ROI is determined and selected by pathologist's guide, and drawn with circle (maximum 660um radius), rectangle (maximum 660x785um) or polygonal shape (maximum 660x785um).
TNP TMA slide contains total 88 cores.
Due to the limitation of scan area in the slide loading slot, only 44 cores per slide can be scanned and collected.
Two TMA slides (88 cores were embedded and slightly shifted to either left or right side of slide to cover half of 88
cores in each slide) are required to collect all cores.
30m
In segment menu, 3 segmentation classes (Immune, Tumor and Stroma) are added and parameters are set in the following order:
For Immune segmentation, Alexa 594 (CD45) is set to positive ("+") and others set to ignore ("0") for the first collection
For Tumor segmentation, Alexa 532 (PanCK) is set to positive ("+") and others set to ignore ("0") for the second collection
For Stroma segmentation, FITC 525 (SYTO 13) is set to positive ("+") and others are set to ignore ("0") for the third collection
Then click Generate Segments.
10m
Once all segments are automatically generated, each channel parameter needs to be manually re-adjusted with pathologist's input to confirm if the segmentation is correctly done.
Caution: less than 20 cells in each segment is removed from collection due to threshold for low signal.
1h 30m
Once all AOI segmentation is complete, Exit Scan Workspace button icon is clicked to approve ROI selection and samples are collected in 96-well plate.
3h
Hybridization
Hybridization
18h 10m
18h 10m
Hybridization
After completion of sample collection, the 96-well plate is finalized, removed from DSP and transferred to the PCR thermocycler.
DNA oligo samples in 96-well plate are completely dehydrated at 60 °C for 01:00:00 in PCR thermocycler.
Caution: the plate lid of PCR thermocycler should be opened completely to avoid the contamination of DNA oligo from evaporation during this dry step.
1h
Add 10 µL of DEPC-treated water to each well and resuspend the DNA oligo samples for 00:30:00 at RT.
30m
(1) Add and mix well 12 µL of each Probe A per panel to a 1.5ml Eppendorf tube (labeled Probe A tube)
Since 7 commercial panels and 1 custom panel (consists of 2 Probe A ) are used, the, total volume is 108 µL (= 12ul x 9 Probe A) in the Probe A tube.
(2) Add and mix well 12 µL of Probe B (Universal probe) with 87 µL of DEPC water in a fresh 1.5ml Eppendorf tube (labeled Probe B tube, total volume is 99 µL ).
(3) Calculate and add 80 µL of hybridization buffer per row into a fresh 1.5ml Eppendorf tube (labeled Master mix tube). For 44 core samples, we collected a total 8 rows in the collection plate so total (= 80 x 8 rows) 640 µL of hybridization buffer was added.
The Probe/Master Mix is made by adding 64 µL from the Probe A tube and 64 µL from the Probe B into the Master mix tube ( 64ul + 64ul + 640ul).
10m
The Hybridization CodeSet for each row (A-H) is taken out from -80 °C and thawed at RT
84 µL of Probe / Master mix is added to each hybridization Codeset and mixed well.
10m
Prepare the hybridization plate separately (96-well plate)
8 µL of hybridization CodeSet mix was added to each well per each row in the hybridization plate.
3 µL of DNA oligo samples from the rehydrated plate (out of 10ul) was mixed into each hybridization Codeset (8ul + 3ul = total 11ul / well).
20m
Seal the hybridization plate with aluminum foil using the microplate heat sealer and briefly spin it down in a centrifuge at 2000g.
Samples are hybridized at 67 °C for 16:00:00 in PCR Thermocycler and then kept at 4 °C until loading in nCounter MAX system.
16h
nCounter reading
nCounter reading
5h 45m
5h 45m
nCounter preparation and reading
In the DSP server, the collection plate is finalized and the library preparation file for nCounter loading is downloaded.
The sample loading volume is determined (it will vary) according to the area size calculation of total ROI/AOI in each column.
The Samples from each row (A-H) are collected and transferred into a 12-well strip PCR tube using a 12-channel multi-pipette.
15m
Briefly, the samples are spun down in the 12-well PCR strip and then transferred to Nanostring's MAX Prep-station to load samples onto the cartridge with the standard sensitivity setting.
3h
The cartridge is transferred to nCounter to read the counts with defined CDF (CodeSet Design Form) setting downloaded from the DSP plate information.
2h 30m
The RCC (Reporter Code Count) file from nCounter reading is downloaded to your PC and imported into the DSP server.
DSP data analysis
DSP data analysis
QC DSP data and analysis
Select and queue the slides to analyze using "New Analysis" in the DSP server.
Determine the New Analysis file name and save it in the designated folder.
Open an analysis file and perform the QC with preset parameters.
QC passed samples are processed and the QC file (CSV) is downloaded to a PC for further analysis.
Comment: All documents related with GeoMx DSP run can be found at https://university.nanostring.com/page/document-library