Oct 25, 2024
NanoString nCounter Gene Expression CodeSet RNA Hybridization Protocol
- 1University of Florida
Protocol Citation: Mackenzie Bolen, Malu G Tansey 2024. NanoString nCounter Gene Expression CodeSet RNA Hybridization Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8drqrg2w/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: October 25, 2024
Last Modified: October 25, 2024
Protocol Integer ID: 110952
Keywords: ASAPCRN, NanoString, nCounter
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020527
Abstract
NanoString’s patented molecular barcodes provide a true digital detection technology capable of highly
multiplexed analysis. CodeSet chemistry uses a Reporter CodeSet and a Capture ProbeSet to bind to the
target. Excess probes are removed, and hybridized probes bind to the cartridge. The target-probe
complexes are immobilized and aligned on the cartridge, and the barcodes are counted on nCounter‱
systems.
Attachments
![Icon for MAN-10056-05-Gene-Expression-Hybridization-Protocol[66].pdf](https://www.protocols.io/img/new-extensions/pdf.svg)
Guidelines
This manual includes instructions for setting up hybridization reactions for gene expression assays that
utilize NanoString’s standard CodeSet chemistry for off-the-shelf XT panels as well as custom-designed
XT assays.
Materials
Items provided by NanoString:
*item, Description, Storage*
1. Reporter CodeSet, Barcoded Reporter Probes, At or below -80°C
2. Capture ProbeSet, Capture Probes, At or below -80°C
3. Reporter Plus CodeSet (optional), Barcoded Reporter Plus Probes, At or below -80°C
4. Capture Plus ProbeSet (optional), Capture Plus Probes, At or below -80°C
5. Hybridization Buffer, Supplied with nCounter Master Kits and SPRINT Reagent Packs, RT (15–25°C)
6. Panel Standard (Optional), Optional sample used for Calibration, At or below -20°C
Additional materials required but not provided by NanoString:
*Item, Manufacturer, Part number*
1. Thermal Cycler, Various, Various
2. Microfuge or picofuge, Various, Various
3. NanoDrop or QuBit*, ThermoFisher, Various
4. Bioanalyzer 2100*, Agilent, G2939BA
5. 12-tube PCR hybridization strip, Various, Various
6. Multi-channel pipettor, Various, Various
7. Pipettes for 0.5–10, 2–20, 20–200 μL*, Rainin, Various
8. 96-well clear polystyrene round-bottom plates*, Corning, 351177
9. Disposable gloves, Various, Various
10. RNeasy‱ Mini Kit* (optional), QIAGEN 74104, 74106
11. Proteinase K Solution (20 mg/mL)**, ThermoFisher, AM2546
nCounter Hybridization
nCounter Hybridization
Pre-heat the thermal cycler to 65°C with a heated lid at 70°C.
NOTE: If you are using cell lysates, see MAN-10051, Preparing RNA and Lysates from Fresh
Frozen Samples.
NOTE: If you are not using Panel Plus or CodeSet Plus, use the CodeSet Hybridization
Setup.
Remove Reporter CodeSet, Capture ProbeSet, Reporter Plus and Capture Plus tubes from the freezer
and thaw at room temperature. Invert or flick the tube several times to mix well and briefly spin down
reagents.
IMPORTANT: After it has thawed, inspect the tube of Reporter CodeSet to make sure no
colored precipitate is present. If you see a colored precipitate, heat the entire tube to
75°C for 10 minutes and cool at room temperature before using.
Create a hybridization master mix by adding the following reagents to the tube containing the
Reporter CodeSet (Table 5). Do not remove the Reporter CodeSet from the tube, add components
directly into the CodeSet tube. Do not add the Capture ProbeSet or Capture Plus to the master mix.
IMPORTANT: If using crude whole cell lysates as sample input, add Proteinase K to the
CodeSet Master Mix at a final concentration of 200 μg/mL (for 20 mg/ml Proteinase K
solution in the final hybridizatiation volume of 18 μl, add 2.5 μL into the 14-reaction
hybridization Master Mix).
Flick or invert the tube repeatedly to mix then briefly spin down the Master Mix.
Label a 12-tube PCR hybridization strip. If necessary, ensure the strip will fit in a microfuge or picofuge
by cutting both the strip tube and its lid in half prior to setting up the reactions, taking care not to
crack the tubes.
Prepare hybridization reactions using a fresh tip for pipetting into each well:
Add 10 μL of Master Mix to each well of a strip tube.
Add 5 µL of sample (or diluted Panel Standard) to each tube containing Master Mix. If you are
using a Panel Standard, see Panel Standard Usage for details.
Mix the Capture ProbeSet and Capture Plus tubes by inverting or flicking, and briefly spin down
the contents.
Create a Master Capture by adding 14 µL of the Capture Plus directly into the Core Capture
ProbeSet tube containing 28 µL. Mix by inverting or flicking, and briefly spin down the contents.
Add 3 µL of Master Capture to each tube. NOTE: Final hyb volume for Core CodeSet + Panel/CodeSet Plus will be 18 µL.
Cap the strip tubes tightly and mix by inverting the tubes several times and flicking to ensure
complete mixing.
Spin briefly and immediately place the tubes in a pre-heated 65°C thermal cycler.
Incubate hybridization reactions for at least 16 hours. Maximum hybridization time should not
exceed 48 hours.
Incubate at 4°C once desired hyb time is reached and process the following day. Do not leave the
reactions at 4°C for more than 24 hours or increased background may result.
NOTE: Counts continue to accumulate with time at 65°C, with total counts typically
increasing 5% per hour between 16 and 24 hours. Although a 16-hour incubation is
adequate for most purposes, a longer incubation increases sensitivity by increasing
counts without significantly increasing background.
Once the hybridization reactions have been removed from the thermal cycler, proceed immediately
to an nCounter Prep Station or SPRINT as described in the nCounter Analysis System User Manual
(MAN-C0035) or nCounter SPRINT User Manual (MAN-10017).
Preparing the RLF
Preparing the RLF
All nCounter Plus reagents are accompanied by an add-in library file (ALF), which specifies the association between each Plus reagent and its target. Information from the ALF must be merged with the reporter library file (RLF) from the original CodeSet prior to scanning on the Digital Analyzer. Failure to merge an ALF with the original RLF will result in no count information being collected for targets of Plus reagents.
Counts that are not scanned are not recoverable on MAX/FLEX instruments. To obtain a merged RLF file, email NanoString at bioinformatics@nanostring.com. Include both the ALF for your Plus product and the RLF for the CodeSet that you want to spike into the Plus product. A new merged RLF will be generated and emailed to the requestor’s address that contains all probe information for both the Plus product and the original CodeSet.
IMPORTANT: Failure to use the merged RLF during the subsequent scan will result in
incomplete barcode scanning and the loss of data from a subset of the included probes.
Protocol references
NanoString nCounter Gene expression protocol identifier: MAN-10056-05-Gene-Expression-Hybridization-Protocol