Feb 07, 2024
Sinai SCENT TMC - Olink Assay using Proseek® Multiplex 96x96 Kit (Juno Biomark System)
- Hui Xie1,
- Manishkumar Patel1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Hui Xie, Manishkumar Patel 2024. Sinai SCENT TMC - Olink Assay using Proseek® Multiplex 96x96 Kit (Juno Biomark System). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3d5zg8j/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: February 06, 2024
Last Modified: February 07, 2024
Protocol Integer ID: 94732
Keywords: protein biomarker, biomarker, proximity extension assay technology, respective protein targets in the sample, juno biomark system, respective protein target, labeled antibody pair, proseek reagent, pcr reporter sequence, oligonucleotide, antibody pair, assay, dependent dna polymerization event, sinai scent tmc, protein, pcr
Abstract
To describe the measurement procedure of 92 protein biomarkers in 1 μL sample volume through Proseek® Multiplex 96×96 Olink. The Proseek reagents are based on PEA, a Proximity Extension Assay technology, in which 96 oligonucleotide-labeled antibody pairs are allowed to bind to their respective protein targets in the sample. A PCR reporter sequence is formed by a proximity-dependent DNA polymerization event and is subsequently detected and quantified using real-time PCR. The assay is performed in a homogeneous 96-well format with no need for washing steps.
Guidelines
Note: Every panel has different Sample Preparation requirement for different sample matrices. Refer to the Step 1. For further details refer to the kit and biological sample specifics before commencing the assay.
Materials
- Proseek multiplex probe kit 96×96 (store at 4 °C )
- Proseek multiplex detection kit 96×96 (store at -20 °C )
- Proseek multiplex controls (store at -20 °C )
- Pipettes and Pipette filter tips
- 96 Well Multiply-PCR Plate half skirt
- Microcentrifuge tubes and FalconTM 15mL Conical Centrifuge Tubes
- 8-well strips tube with lids
- Multi-channel pipette reservoir
- Adhesive plastic film (heat-resistant)
- High purity water (sterile filtered, MilliQ or similar)
- Microcentrifuge for tubes and Centrifuge for plates
- Vortex
- VWR Reagent reservior 25ml
- Freezing block (-20 °C ) for enzyme handling
- Refrigerator or cold room (2 °C to 8 °C )
- Bio-RAD T100 Thermal Cycle
- Fluidigm BioMarkTM HD System
- Juno System
Before start
- Wear protective gloves and lab coat while performing this procedure.
- Perform all open-vessel work within a laminar flow biological safety cabinet to avoid contamination of samples or operator exposure to potentially infectious materials.
- Dispose of all biological waste within the appropriate waste containers.
Sample preparation Notes
The serum and plasma samples for the Olink Inflammation and Immuno-Oncology panel are run neatly. However, every panel has different Sample Preparation requirements for different sample matrices. For example:
- Serum and Plasma samples for the Olink CVD III panel should be diluted 1:100 using the Olink sample diluent.
- Human Breast Milk samples for the Olink Inflammation panel should be diluted 1:2 with Olink sample diluent. The samples must be prepared following the SOP HIMC-4042: PREANALYTICAL PROCEDURE OF HUMAN BREAST MILK SAMPLES FOR OLINK ASSAY.
Overview of the assay
Day 1: Sample incubation with Probes
16h 5m 10s
Important Note: All the vortex steps require 00:00:30 vortex time, except for the Enzyme and PCR polymerase.
30s
Thaw samples, vortex and spin down at 400 x g, Room temperature, 00:01:00
1m
Thaw the Incubation Stabilizer from the Proseek Multiplex Controls box at room temperature, vortex for 00:00:30 and spin briefly, then place On ice .
30s
Thaw the interplate control (IPC) and negative control from the Proseek multiplex controls
box in Room temperature , vortex 00:00:30 and spin briefly, place On ice .
Prepare an 8-strip tube and mark the negative control on the top. Add 3x5 μL Negative Control and 3x5 μL IPC in an 8-strip tube (Figure 1).
30s
Prepare incubation mix in 1.5mL microcentrifuge tube, vortex 00:00:30 and spin each reagent before transfer to the mix, pipette the incubation solution carefully to avoid foaming.
30s
Vortex the incubation mix for 00:00:30 and spin down the content. Transfer 47 µL per well of the Incubation mix to a new 8-well strip. Vortex the strip tube for 00:00:10 and spin down.
40s
Prepare a 96-well plate and label as Incubation plate. Mark the control position on the plate according to plate layout (Figure 2).Use a multi-channel pipette to transfer 3 µL of the Incubation mix from the 8-well strip to the bottom of each well of a 96-well plate by using reverse pipetting. Pipette from the uppermost part of the Incubation mix to prevent liquid from sticking to the outside of the pipette tip. Do not change pipette tips.
Use amulti-channel pipette to add 1 µL of each sample to the bottom of thesample well of the
incubation plate, according to the plate layout in Figure 2.
Use a multi-channel pipette to add 1 µL of negative control and IPC from 8-strip to the bottom of the well in position C12-H12, according to the plate layout in Figure2.
Seal the incubation plate with an adhesive plastic film. It is important that all wells are properly sealed, especially around the edges, to avoid evaporation of samples. Vortex the incubation plate for 00:00:30 and Spin down the content at 400 x g, Room temperature, 00:01:00
1m 30s
Incubate the Incubation Plate overnight at 2 °C to 8 °C . Be consistent for the incubation time at 16:00:00
16h
Day 2: Extension of the Conjugated Oligonucleotide
1h 48m 40s
Turn on your thermal cycler (HIMC Bio-Rad T100 Thermal Cycle) and create a PEA (Proximity Extension Assay technology) program on the thermal cycler with the following conditions:
Preheat the PCR machine by running this protocol and pause when the sample temperature reaches to 50 °C
Thaw the PEA Solution, vortex, and spin briefly. Prepare the following extension mix in a 15ml Falcon tube. Use an ice box when removing the PEA Enzyme and the PCR Polymerase from -20 °C . Vortex 00:00:10 and spin down the content briefly before pipetting the enzymes into the mix. Keep the leftover of the PCR polymerase in the ice box, which will be used for the further step.
10s
Vortex the Extension mix for 00:00:30
30s
Bring the Incubation Plate to room temperature and spin down at 400 x g, Room temperature, 00:01:00
1m
Pour the Extension mix into a multi-channel pipette reservoir.
Carefully remove the plastic adhesive film from the Incubation Plate.
Steps 20 - 23 have to be performed within 00:05:00 .
Start a timer before transferring 96 µL of Extension mix to each well of the Incubation Plate by using reverse pipetting. Do not change pipette tips, place the tips against the upper parts of the well wall.
5m
Make sure the tips never come in contact with the contents of the wells.
Add a new aluminum seal film to the Incubation Plate; It is important that all wells are properly sealed, especially around the edges to avoid evaporation of samples
Vortex the plate thoroughly for at least 00:00:30 and ensure that all wells are mixed. Spin down the content at 400 x g, Room temperature, 00:01:00
1m 30s
Stop the PCR machine which was paused before, and wait the sample temperature to cool down to 30 °C . After the 00:05:00 timer done for previous step, place the Incubation Plate in the preheated thermal cycler and run the PEA program. This program will be approximately 01:40:00
1h 45m
Continue with the Detection step or store the Incubation Plate for up to one week at 4 °C
Detection of targeted oligos throughout real-time PCR
4h 25m 20s
Turn on the “Juno System” and verify that the HX interface plate has been correctly installed. Otherwise, change the Interface plate according to the following instruction: Tap “Tools” and “interface plate” bottom in the screen and select “Switch interface plate”. Place the HX interface plate and the loading fixture on the tray and tap “Install”. After the interface plate is installed, remove the loading fixture and tap “Close”
Take a new 96.96 Dynamic Array IFC. Inject control line fluid into each accumulator on the chip. Remove and discard the black protective film from the bottom to the chip. Place the chip into the IFC controller then run the “Primer” script. The Primer program will takes approximately 00:20:00 . Chip needs to be loaded within 00:30:00 after completed Primer, otherwise chip need to be primer again
50m
Thaw the Primer Plate (offered in the kit) at 0 °C , Vortex the plate for Room temperature after thawing.
Thaw the Detection Solution, vortex 00:00:30 and spin briefly; prepare the following Detection mix in a 1.5ml microcentrifuge tube; vortex 00:00:10 and spin down Detection Enzyme and PCR Polymerase briefly before pipetting the enzymes into the mix.
40s
Vortex 00:00:30 of the Detection mix and spin briefly; transfer 95 µL of the Detection mix per well to an 8-well strip. Vortex the strip tube for 00:00:10 and spin briefly.
40s
Label a new 96-well plate as “Sample Plate”; use amulti-channel pipette to transfer 7.2 µL of Detection mix to each well of a new 96-well plate by reverse pipetting.
Remove the Incubation Plate from the thermal cycler; vortex the plate for 00:00:30 and spin down the contents.
30s
Carefully remove the plastic film and transfer 2.8 µL from each well of the Incubation Plate to the Sample Plate.
Seal the Sample Plate with a new plastic adhesive film, vortex the plate for 00:00:30 and spin with Primer Plate at 400 x g, Room temperature, 00:01:00
1m 30s
Gently remove the Primer Plate sealing to avoid contamination between wells; transfer 5 µL from each well of the Primer Plate to the primed 96.96 Dynamic Array IFC by using reverse pipetting; change pipette tips after each primer; Primers are loaded into their respective inlets on the left side of the chip according to Figure 3.
Transfer 5 µL from each well of the Sample Plate into the inlets on the right side of the chip according to Figure 3 by reverse pipetting; change pipette tips after each sample; See Figure 3 for a detailed instruction on sample loading.
Remove any visible bubbles, e.g., with a syringe needle.
After the samples and primers are loaded in all wells, place the chip with barcoding facing you in the IFC Controller for loading. Select “load” and “load mix 96.96 GE IFC” followed by “Run Script” to load the assay and sample mixes into the central portion of the Dynamic array. The Program will take approximately 01:32:00
1h 32m
Press the on/off button (on the right side of the instrument) to turn on the Biomarker and press the round button (on the left side) to connect the computer.
Open the “Biomarker Data Collection” and select the “start a new run” on the main menu, the lid will open and the tray come out on the Biomarker.
After the “Load mix 96.96 GE IFC” program is done, “Eject” the chip from the “Juno” system and remove the tiny dust from the Central Portion using the cello-tape, do this even though no visible dust. Take a piece of tape and let the sticky part touch (do not press down) the surface of the chip.
Load the IFC chip into the “Biomarker” tray with the barcode facing outwards and Select “Load”, the chip is now loaded into the instrument.
Select “Next” once the barcode and chip type is read and visible in next page. Select the “This is a new chip run” on this page and name the plate using HIMC format Then press “Next”
Set the following options for application reference and probe setting according to Figure 4.
Please select the following:
- Application: Gene Expression
- Passive Reference: ROX
- Assay: Single Probe
- Probes: FAM-MGB
Then select “Next”, and start run.
Select the “Olink_PE_96X96_New_35_03072019.pcl” protocol from the Olink folder on the desktop. Verify the protocol according to Figure 5. Then “Next”
Press “Start run” to start the program. The PE Program will take approximately 02:00:00 . There will be a message on screen “The Run has been successfully completed” after Run is finished.
2h
“Eject” the Chip from the Biomarker instrument.
Use an OLINK assay dedicated Flash Drive, and copy the study whole Folder with the Run name on the Flash Drive. Eject the flash drive.
If not running any more Chips on Bio-Mark on the same day, close the Bio-Mark software, shut down the Computer, and turn off the Bio-Mark machine power button.
Follow the SOP “HIMC-4031_R2_OlinkPEA_DataAnalysis”.
Protocol references
REFERENCE-Proseek® Multiplex 96×96 Kit, User Manual (http://www.olink.com/products/document-download-center/)