PAXgene Processing by RNA Extraction

Place all 2 PAXgene tubes in the 4°C fridge from blood draw if RNA extraction is NOT to be done the next day.

Incubate PAXgene Blood RNA Tubes for 24h 0m 0s at 4Room temperature (25°C) after blood collection or removal from 4°C before processing.

Prepare 55°C and 65°C heating blocks.

Centrifuge tubes at 4000rpm,25°C.

Remove supernatant and add 4mL to each tube.

Close tube using a fresh secondary Hemogard closure.

Vortex 0h 0m 10s until pellet is dissolved.

Centrifuge at 4000rpm,25°C. Discard supernatant.

Add 350µL, cap and vortex until the pellet is visibly dissolved.

Pipette sample into a 1.5 ml microcentrifuge tube.

Add 300µL and 40µL.

Mix by votexing briefly, then incubate for a total of 10 minutes on a heating block at 55°C as follows: incubate for 0h 5m 0s, briefly vortex, incubate 0h 5m 0s.

Pipette lysate directly onto the membrane of a PAXgene Shredder spin column (lilac-colored) placed in a 2 ml processing tube.

Centrifuge at 13000rpm,25°C.

Carefully pipette supernatant of the flow-through to a 1.5 microcentrifuge tube without disturbing pellet.

Add 350µL, mix by votexing, and centrifuge 1000x g to remove drops from inside of the tube lid.

Pipette 700µL into the PAXgene RNA spin column (pink) placed in a 2 ml processing tube, and centrifuge at 10000rpm,25°C.

Place the spin column in a new 2 ml processing tube.

Pipette the remaining sample from step 16 into the spin column and centrifuge at 10000rpm,25°C.

Place spin column in a new 2 ml processing tube.

Add 350µL into the spin column and centrifuge at 10000rpm,25°C.

Place spin column in a new 2 ml processing tube and discard old tube.

Add 10µL to 70µL in a 1.5 ml microcentrifuge tube and mix by gently flicking the tube (do not vortex). Centrifuge briefly to collect residual liquid on sides.

Add 80µL directly onto the membrane of the spin column, and place on benchtop (20°C - 30°C, 55Room temperature) for 0h 5m 0s.

Pipette 350µL into the spin column and centrifuge at 10000rpm,25°C.

Place spin column in a new 2 ml processing tube and discard old tube.

Add 500µL (diluted in 1:4 in 100% ethanol) into the spin column, and centrifuge at 10000rpm,25°C.

Place spin column in a new 2 ml processing tube and discard old tube.

Add another 500µL to the spin column and centrifuge at 10000rpm,25°C.

Place spin column into a new 2 ml processing tube and discard old tube. Centrifuge at 13000rpm,25°C.

Place spin column into a new 1.5 ml microcentrifuge tube and discard old tube.

Add 41µL directly onto spin column membrane and sit for 0h 1m 0s.

Centrifuge for 10000rpm,25°C,0h 0m 0s to elute the RNA.

Repeat step 32 with 41µL and the same microcentrifuge tube: sit for 0h 1m 0s. Centrifuge for 10000rpm,25°C,0h 0m 0s to elute the RNA.

Combine all two elutes into one tube. Split volume in half between two tubes and label (RNA-01 and 02).

Place the aliquots in the 65°C heat block for 0h 5m 0s without shaking. After incubation, chill immediately 65On ice.

Aliquot 3µL into a 1.5 mL tube for Nanodrop concentration assay.

Aliquot 2µL into a PCR tube for Agilent 2100 Bioanalyzer assay. (Store in -20°C if not being assay immediately.)

Scan and position RNA in the Freezerworks Inventory Program.

Store in corresponding -80°C freezer.