Extracting RNA from PAXgene fixed tissue, PAXgene fixed & Embedded Tissue, and Fresh Tissue

Protocol: Qiagen RNeasy Kit

Notes:

• β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µl β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing.
• Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution.

Procedure:

1. Make sure all buffers are prepared appropriately
2. Label tubes + clean bench
3. Tissue prep steps
   1. For fresh or fresh-frozen tissue
      1. Place tissue in up to 600 µl Buffer RLT with BME added (600 + 6 uL)
      2. Disrupt the tissue and homogenize the lysate in Buffer RLT
         1. I am going to try normal bead-beating like we do for the DNA/RNA shield samples
         2. Alternatives: tissue lyser, rotor-star homogenizer
   2. For cryosections:
      1. Pre-cool tube with 150 uL buffer RLT (with BME = 150 + 1.5 uL) + 290 uL water + 10 uL ProK
         1. Final vol = 450
      2. Add 1-3 cryosections + vortex to mix
      3. Incubate at 56 ºC for 15 min at 1400 rpm
      4. Proceed to step 4
         1. potential use of qiashredder here or other method such as bead-beating to homogenize completely
   3. For PAXgene-fixed tissues in PAXgene stabilizer:
      1. Place tissue in 250 µl Buffer RLT with BME added (250 + 1.5 uL)
      2. Disrupt the tissue and homogenize the lysate in Buffer RLT
         1. I am going to try normal bead-beating like we do for the DNA/RNA shield samples
         2. Alternatives: tissue lyser, rotor-star homogenizer
      3. Add 480 µl RNase-free water to cell lysate suspension. Then add 20 µl Proteinase K and mix by vortexing for 5 s.
         1. Final vol = 750
      4. Incubate at 45 ºC for 15 min at 1400 rpm
      5. Proceed to step 4
4. Centrifuge the lysate for 3 min at full speed.
   1. Carefully remove the supernatant by pipetting, and transfer it to a new microcentrifuge tube (not supplied). Use only this supernatant (lysate) in subsequent steps.
5. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately by pipetting.
6. Transfer up to 700 µl of the sample to an RNeasy spin column placed in a 2 ml collection tube (supplied).
   1. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.
   2. Reuse the collection tube in step 7.
   3. If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.
7. Add 700 µl Buffer RW1 to the RNeasy spin column.
   1. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
   2. Reuse the collection tube in step 8.
8. Add 500 µl Buffer RPE to the RNeasy spin column.
   1. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
   2. Reuse the collection tube in step 9.
   3. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Things to do before starting”).
9. Add 500 µl Buffer RPE to the RNeasy spin column.
   1. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash + dry the spin column membrane.
10. Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through.
    1. Close the lid gently, and centrifuge at full speed for 1 min.
11. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50 µl RNAse-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.

RNA Quality Check: Tapestation

Full results can be found here