TITLE: QuantiGene multiplex assay
AUTHORS: Amy R Hicks

[DESCRIPTION]
This protocol outlines a method of simultaneously measuring the expression of up to 80 genes in any one sample using the QuantiGene multiplex assay from ThermoFisher. This protocol was adapted from the lab of Professor Gill Bates. Cells are grown in 96-well plates, lysed, snap frozen and stored at -80oC. Lysed samples are then incubated overnight with magnetic capture beads as well as a probe panel to detect a custom designed set of genes. A series of incubations and washes amplify the captured RNA signals, which are subsequently measured using a fluorescent probe on a Magpix (Luminex). 

[STEPS]
SECTION: Sample preparation
1. Pre-warm lysis mixture at 37oC for 30 mins, followed by gentle swirling.30 min 

SECTION: Sample preparation
2. Prepare working lysis mixture by adding 10 µL  of proteinase K to each 1 mL  of lysis mixture required.

SECTION: Sample preparation
3. Add 1/2 volume of working lysis mixture to cells in culture media from a reagent reservoir using the multichannel pipette (e.g. for a 96 well plate containing 100 µL of media per well, add 50 µL of working lysis mixture).

SECTION: Sample preparation
4. Mix by pipetting up and down 3-4 times, discard the tips before continuing with the consecutive wells (more is more).
SECTION: Sample preparation
5. Snap freeze on a bed of dry ice and store in the -80°C until required.
SECTION: Sample preparation
6. When required, incubate the cell culture plate in the Vortemp pre-warmed to 50°C for 60 min without shaking.

SECTION: Sample preparation
7. Verify cell lysis using the cell culture microscope.
SECTION: Sample preparation
7.1. For new QuantiGene plexes, appropriate dilutions must be ascertained via a serial dilution experiment- refer to Papadopoulou et al, 2019 and pages 29–30 of the QuantiGene Plex Gene Expression Assay User Guide for more details.
SECTION: Storage of lysates
8. Samples should be stored long term in the -80°C freezer. Samples do not need to be thawed on ice and are stable at RT.
SECTION: Assay day 1
9.   Pre-warm lysis mixture at 37°C for 30 min followed by gentle swirling.

SECTION: Assay day 1
10. Arrange sufficient pre-vortexed and diluted samples (for experiment) or serial dilutions and reference RNA (for plex optimisation) as per your plate plan and keep at RT- each 40 µL sample should be run in duplicate, include a background control by making sufficient diluted lysis mixture (1 volume lysis mixture plus 2 volumes of RNase-free water).

SECTION: Assay day 1
11. Handle the reagents listed below as follows:
SECTION: Assay day 1
12. Prepare an appropriate volume of working bead mix by combining the following reagents in the order listed (this is for 2- to 64-plex assays), scale according to the number of wells on your QuantiGene plate(s), keep working bead mix at RT and protected from light.
SECTION: Assay day 1
13. Vortex Working Bead Mix for 10 seconds and then carefully pipette 10μl into each well of a magnetic separation plate, avoiding bubbles, add 40 µL of each sample (including background controls) as per your plate plan into the magnetic separation plate (load each sample with a new pipette tip).

SECTION: Assay day 1
14. Seal magnetic separation plate with a pressure seal. Use the backing of the pressure seal to firmly and evenly apply pressure across the whole seal and lastly run your finger along each edge of the plate to seal.
SECTION: Assay day 1
11.1. Probe Set & Blocking Reagent (kept at -20°C in QuantiGene reagents box): thaw and vortex briefly to mix, then centrifuge probe set briefly to collect contents at the bottom of the tube.
SECTION: Assay day 1
11.2. Proteinase K (kept at -20°C in QuantiGene reagents box): keep on ice. 
SECTION: Assay day 1
11.3. Capture Beads (kept at 4°C in QuantiGene capture bead box): take out of storage right before use and protect from light. 
SECTION: Assay day 1
12.1. 
OrderReagent1 well (μl)96 wells (+14 for extra) (μl)1Nuclease-free water2.62862Lysis Mixture3.33633Blocking Reagent11104Proteinase K0.1115Capture Beads (vortex for 30 seconds before adding)0.5556Probe Set2.5275TOTAL:101100

 
 
SECTION: Assay day 2
16. Turn on Magpix and computer to allow lasers time to warm up.
SECTION: Assay day 2
17. Warm pre-amplifier solution, amplifier solution, label probe solution and SAPE diluent at 37°C at least 30 min prior to use.

SECTION: Assay day 2
18. Prepare 1 x wash buffer by adding 3 mL wash buffer component 1 and 50 mL wash buffer component 2 and topping up to 1 L with nuclease-free water from the Milli-Q or Hyclone water.

SECTION: Assay day 2
19. Remove the magnetic separation plate from the shaking incubator and adjust temperature to 51°C at 600rpm.
SECTION: Assay day 2
20. Centrifuge magnetic separation plate at 240 × g for 1 min at RT.

SECTION: Assay day 2
21. In the fume hood, insert magnetic separation plate into handheld magnetic plate washer and ensure it is securely locked, allow 1 min to allow magnetic beads to accumulate on bottom of each well.

SECTION: Assay day 2
22. Keep plate inserted in handheld magnetic plate washer at all times for this step: add 100 µL of 1 x wash buffer, wait 15 s to allow the magnetic beads to accumulate at the bottom of each well, remove solution by quickly inverting over a waste container and gently blot on several layers of paper towel to remove residual solution.

SECTION: Assay day 2
23. Repeat previous step two more times.
SECTION: Assay day 2
24. Add 50 µL of pre-amplifier solution to each well and seal with a foil plate seal, return to Vortemp and incubate for 60 min at 51°C at 600rpm (the minimum time for incubation is 45 min and maximum is 120 min).

SECTION: Assay day 2
25. Repeat steps 21-24 for amplifier solution in place of pre-amplifier solution.
SECTION: Assay day 2
26. Repeat steps 21-24 for label probe solution in place of pre-amplifier solution.
SECTION: Assay day 2
27. Prepare SAPE working reagent by mixing 3 µL of SAPE to 1 mL SAPE diluent (scaled to the number of the wells on your plates + 10%), vortex to mix and keep at RT protected from light.

SECTION: Assay day 2
28. Repeat steps 21-24 then add 50 µL of SAPE working reagent to each well and seal with a foil plate deal, return to Vortemp and incubate for 30 min at 51°C at 600 rpm before turning off Vortemp. Do not exceed 30 min incubation.

SECTION: Assay day 2
29. Repeat steps 21-24 with SAPE wash buffer in place of regular wash buffer.
SECTION: Assay day 2
30. To prepare the plate for analysis, add 130 µL of SAPE wash buffer to each well, seal the plate with a foil plate seal, tape sealed magnetic separation plate down onto a shaker and shake vigorously (~800rpm) at RT, immediately run plate on Magpix.

SECTION: Assay day 2
31. If analysing more than one plate, keep consecutive plates at room temperature protected from light until required and then prepare as in step 32 and shake vigorously (~800rpm) at RT before analysing on the Magpix instrument.
SECTION: Assay day 2
32. Plates can be stored long-term in at 4°C, for reanalysis of stored plates, repeat steps 29-30.
SECTION: Assay day 1
15. Place the magnetic separation plate in the Vortemp shaking incubator for 1080 min to 1320 min at 54°C at 600rpm.