QuantiGene multiplex assay

Pre-warm lysis mixture at 37oC ^oC for 30 mins , followed by gentle swirling.0h 30m 0s

Prepare working lysis mixture by adding 10µL of proteinase K to each 1mL of lysis mixture required.

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 100µL of media per well, add 50µL of working lysis mixture).

Mix by pipetting up and down 3-4 times, discard the tips before continuing with the consecutive wells (more is more).

Snap freeze on a bed of dry ice and store in the -80°C until required.

When required, incubate the cell culture plate in the Vortemp pre-warmed to 50°C for 1h 0m 0s without shaking.

Verify cell lysis using the cell culture microscope.

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.

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.

µL Pre-warm lysis mixture at 37°C for 0h 30m 0s followed by gentle swirling.

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 40µL 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).

Handle the reagents listed below as follows:

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.

Proteinase K (kept at - 20°C in QuantiGene reagents box): keep on ice .

Capture Beads (kept at 4°C in QuantiGene capture bead box): take out of storage right before use and protect from light .

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.

A	B	C	D
Order	Reagent	1 well (μl)	96 wells (+14 for extra) (μl)
1	Nuclease-free water	2.6	286
2	Lysis Mixture	3.3	363
3	Blocking Reagent	1	110
4	Proteinase K	0.1	11
5	Capture Beads (vortex for 30 seconds before adding)	0.5	55
6	Probe Set	2.5	275
TOTAL:	10	1100

Working bead mix

Vortex Working Bead Mix for 10 seconds and then carefully pipette 10μl into each well of a magnetic separation plate , avoiding bubbles, add 40µL of each sample (including background controls) as per your plate plan into the magnetic separation plate (load each sample with a new pipette tip).

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.

Place the magnetic separation plate in the Vortemp shaking incubator for to 18h 0m 0s to 22h 0m 0s at 54°C at 600rpm.

Turn on Magpix and computer to allow lasers time to warm up.

Warm pre-amplifier solution , amplifier solution , label probe solution and SAPE diluent at 37°C at least 0h 30m 0s prior to use.

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

Remove the magnetic separation plate from the shaking incubator and adjust temperature to 51°C at 600rpm .

Centrifuge magnetic separation plate at 240 × g for 0h 1m 0s at RT.

In the fume hood, insert magnetic separation plate into handheld magnetic plate washer and ensure it is securely locked, allow 0h 1m 0s to allow magnetic beads to accumulate on bottom of each well.

Keep plate inserted in handheld magnetic plate washer at all times for this step: add 100µL of 1 x wash buffer , wait 0h 0m 15s 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.

Repeat previous step two more times.

Add 50µL of pre-amplifier solution to each well and seal with a foil plate seal , return to Vortemp and incubate for 1h 0m 0s at 51°C at 600rpm (the minimum time for incubation is 0h 45m 0s and maximum is 2h 0m 0s).

Repeat steps 21-24 for amplifier solution in place of pre - amplifier solution.

Repeat steps 21-24 for label probe solution in place of pre - amplifier solution.

Prepare SAPE working reagent by mixing 3µL of SAPE to SAPE diluent 1mL SAPE diluent (scaled to the number of the wells on your plates + 10% ), vortex to mix and keep at RT protected from light.

Repeat steps 21-24 then add 50µL of SAPE working reagent to each well and seal with a foil plate deal, return to Vortemp and incubate for 0h 30m 0s at 51°C at 600 rpm before turning off Vortemp. Do not exceed incubation. 0h 30m 0s incubation.

Repeat steps 21-24 with SAPE wash buffer in place of regular wash buffer .

To prepare the plate for analysis, add 130µL 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.

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.

Plates can be stored long - term in at 4°C, for reanalysis of stored plates, repeat steps 29-30.