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 3000?5000 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 3000?5000 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 shaker?incubator, 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 (1?2 s; £ 1000 x g ) to remove drops from the inside of the tube lid.

Note: The length of the centrifugation must not exceed 1?2 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).

8.Apply 700 µl sample to the PAXgene column sitting in a 2 ml processing tube, and centrifuge for 1 min at ³ 8000 x g ( ³ 10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.

9.Apply the remaining sample to the PAXgene column, and centrifuge for 1 min at ³ 8000 x g ( ³ 10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.

We do not perform the optional DNAse step.

10.Apply 700 µl Buffer BR3 to the PAXgene column, and centrifuge for 1 min at ³ 8000 x g ( ³ 10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.

11.Apply 500 µl Buffer BR4 to the PAXgene column, and centrifuge for 1 min at ³ 8000 x g ( ³ 10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.

Note: Buffer BR4 is supplied as a concentrate. Ensure that ethanol is added to Buffer BR4 before use (see “Important notes before starting”).

12.Add another 500 µl Buffer BR4 to the PAXgene column. Centrifuge for 3 min at maximum speed to dry the PAXgene column membrane.

After this washing step, the silica-gel membrane may be light or dark brown in color. This does not influence the quality of the RNA isolated.

13.Discard the tube containing the flow-through, and place the PAXgene column in a new 2 ml processing tube (not supplied). Centrifuge for 1 min at full speed.  DO this, NOT optional ? need to be sure to remove all BR4 from sample !

14.To elute, discard the tube containing the flow-through, transfer the PAXgene column to a 1.5 ml elution tube, and pipet 40 µl Buffer BR5 directly onto the PAXgene column membrane. Centrifuge for 1 min at ³ 8000 x g ( ³ 10,000 rpm) .

It is important to wet the whole membrane with Buffer BR5 in order to achieve maximum elution efficiency.  Be careful to add BR5 to middle of membrane, and not to touch membrane with pipet tip.

15.Repeat the elution step (step 14) as described, using 40 µl Buffer BR5.

16.Store samples at ?70℃ or ?80℃.  We store the purified total RNA immediately and denature prior to further manipulation.  However, the official protocol provides the option of denaturing RNA at 65℃ for 10 minutes before storing at -80℃.