TITLE: Sinai SCENT TMC - Olink Assay using Proseek® Multiplex 96x96 Kit (Juno Biomark System)
AUTHORS: Hui Xie, Manishkumar Patel

[DESCRIPTION]
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.

[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.

[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.

[STEPS]
SECTION: Sample preparation Notes
1. 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.
SECTION: Overview of the assay
2. 
 


SECTION: Day 1: Sample incubation with Probes
3. Important Note: All the vortex steps require 30 s vortex time, except for the Enzyme and PCR polymerase.
SECTION: Day 1: Sample incubation with Probes
4. Thaw samples, vortex and spin down at 400 x g, 1 min
SECTION: Day 1: Sample incubation with Probes
5. Thaw the Incubation Stabilizer from the Proseek Multiplex Controls box at room temperature, vortex for 30 s and spin briefly, then place on ice.
SECTION: Day 1: Sample incubation with Probes
6. Thaw the interplate control (IPC) and negative control from the Proseek multiplex controls
box in Room temperature, vortex 30 s 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).


 

SECTION: Day 1: Sample incubation with Probes
7. Prepare incubation mix in 1.5mL microcentrifuge tube, vortex 30 s and spin each reagent before transfer to the mix, pipette the incubation solution carefully to avoid foaming.

 

SECTION: Day 1: Sample incubation with Probes
8. Vortex the incubation mix for 30 s 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 10 s and spin down.
SECTION: Day 1: Sample incubation with Probes
9. 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.


 

SECTION: Day 1: Sample incubation with Probes
10. 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.
SECTION: Day 1: Sample incubation with Probes
11. 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.
SECTION: Day 1: Sample incubation with Probes
12. 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 30 s and Spin down the content at 400 x g, 1 min
SECTION: Day 1: Sample incubation with Probes
13. Incubate the Incubation Plate overnight at 2 °C to 8 °C. Be consistent for the incubation time at 960 min

SECTION: Day 2: Extension of the Conjugated Oligonucleotide
14. 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

 

SECTION: Day 2: Extension of the Conjugated Oligonucleotide
15. 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 10 s 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.

 

SECTION: Day 2: Extension of the Conjugated Oligonucleotide
16. Vortex the Extension mix for 30 s
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
17. Bring the Incubation Plate to room temperature and spin down at 400 x g, 1 min
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
18. Pour the Extension mix into a multi-channel pipette reservoir.
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
19. Carefully remove the plastic adhesive film from the Incubation Plate.
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
20. Steps 20 - 23 have to be performed within 5 min. 

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.
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
21. Make sure the tips never come in contact with the contents of the wells.
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
22. 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
SECTION: Day 2: Extension of the Conjugated Oligonucleotide
23. Vortex the plate thoroughly for at least 30 s and ensure that all wells are mixed. Spin down the content at 400 x g, 1 min

SECTION: Day 2: Extension of the Conjugated Oligonucleotide
24. Stop the PCR machine which was paused before, and wait the sample temperature to cool down to 30 °C. After the 5 min timer done for previous step, place the Incubation Plate in the preheated thermal cycler and run the PEA program. This program will be approximately 100 min

SECTION: Day 2: Extension of the Conjugated Oligonucleotide
25. Continue with the Detection step or store the Incubation Plate for up to one week at 4 °C
SECTION: Detection of targeted oligos throughout real-time PCR
26. 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”
SECTION: Detection of targeted oligos throughout real-time PCR
27. 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 20 min. Chip needs to be loaded within 30 min after completed Primer, otherwise chip need to be primer again

SECTION: Detection of targeted oligos throughout real-time PCR
28. Thaw the Primer Plate (offered in the kit) at °C, Vortex the plate for Room temperature after thawing.
SECTION: Detection of targeted oligos throughout real-time PCR
29. Thaw the Detection Solution, vortex 30 s and spin briefly; prepare the following Detection mix in a 1.5ml microcentrifuge tube; vortex 10 s and spin down Detection Enzyme and PCR Polymerase briefly before pipetting the enzymes into the mix.

 

SECTION: Detection of targeted oligos throughout real-time PCR
30. Vortex 30 s 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 10 s and spin briefly.
SECTION: Detection of targeted oligos throughout real-time PCR
31. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
32. Remove the Incubation Plate from the thermal cycler; vortex the plate for 30 s and spin down the contents.

SECTION: Detection of targeted oligos throughout real-time PCR
33. Carefully remove the plastic film and transfer 2.8 µL from each well of the Incubation Plate to the Sample Plate.
SECTION: Detection of targeted oligos throughout real-time PCR
34. Seal the Sample Plate with a new plastic adhesive film, vortex the plate for 30 s and spin with Primer Plate at 400 x g, 1 min
 

SECTION: Detection of targeted oligos throughout real-time PCR
35. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
36. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
37. Remove any visible bubbles, e.g., with a syringe needle.
SECTION: Detection of targeted oligos throughout real-time PCR
38. 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 92 min

SECTION: Detection of targeted oligos throughout real-time PCR
39. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
40. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
41. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
42. Load the IFC chip into the “Biomarker” tray with the barcode facing outwards and Select “Load”, the chip is now loaded into the instrument.
SECTION: Detection of targeted oligos throughout real-time PCR
43. 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”
SECTION: Detection of targeted oligos throughout real-time PCR
44. 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.
 

SECTION: Detection of targeted oligos throughout real-time PCR
45. 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”

 

SECTION: Detection of targeted oligos throughout real-time PCR
46. Press “Start run” to start the program. The PE Program will take approximately 120 min. There will be a message on screen “The Run has been successfully completed” after Run is finished.
SECTION: Detection of targeted oligos throughout real-time PCR
47. “Eject” the Chip from the Biomarker instrument.
SECTION: Detection of targeted oligos throughout real-time PCR
48. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
49. 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.
SECTION: Detection of targeted oligos throughout real-time PCR
50. Follow the SOP “HIMC-4031_R2_OlinkPEA_DataAnalysis”.