Preparation of total RNA from whole blood collected in PAXgene tubes

Short PAXgene Protocol:

spin in step 6 increased to 20 min; no Dnase treatment; optional wash after step 12 REQUIRED

1.Centrifuge the PAXgene Blood RNA Tube for 10 min at 30005000 x g using a swing-out rotor.

Note: Ensure that the blood sample has been incubated in the PAXgene Blood RNA Tube for a minimum of 2 h at room temperature, in order to achieve complete lysis.

Preheat a water bath or heat block (for microcentrifuge tubes) to 55℃, during first spin.

We spin at 3750 rpm in a standard table top centrifuge.

2.Remove the supernatant by decanting or pipetting. Add 5 ml RNase-free water to the pellet, and close the tube using a fresh secondary Hemogard closure.

Gently decant the supernatant, and blot on a paper towel.

3.Thoroughly resuspend the pellet by vortexing, and centrifuge for 10 min at 30005000 x g . Remove and discard the entire supernatant.

Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure.

Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate which will affect the conditions for binding RNA to the PAXgene membrane.

Pellet does NOT wash to clear/white color; will remain brown.

4.Thoroughly resuspend the pellet in 360 µl Buffer BR1 by vortexing.

5.Pipet the sample into a 1.5 ml or 2 ml microcentrifuge tube (not supplied). Add 300 µl Buffer BR2 and 40 µl Proteinase K. Mix by vortexing, and incubate for 10 min at 55℃ using a shakerincubator, heating block, or water bath.

If a heating block or water bath is used, vortex each sample once during the incubation. Do not allow the temperature of the sample to decrease during vortexing.

Note: Do not mix Buffer BR2 and Proteinase K together before adding them to the sample.

6.Centrifuge for 20 min at maximum speed in a microcentrifuge. Transfer the supernatant to a fresh 1.5 ml or 2 ml microcentrifuge tube (not supplied).

A minimum g-force of 10,000 x g is required.

Transfer of small debris remaining in the supernatant after centrifugation at full speed will not affect the procedure.

7.Add 350 µl 95 % ethanol. Mix by vortexing, and centrifuge briefly (12 s; £ 1000 x g ) to remove drops from the inside of the tube lid.

Note: The length of the centrifugation must not exceed 12 s, as this may result in pelletting of nucleic acids and reduced yields of total RNA.

use 95% ethanol; 100% ethanol often contains benzene (bad for arrays).