NOTEBOOK

Extraction of RNA

Materials needed Qiagen RNeasy mini kit

• 1) Pipette 600 µl blood in a tube of 1.5 mL.
• 2) Add 900 µl of lysis buffer, incubate for 10 minutes under stirring.
• 3) After centrifugation for 5 minutes at 14.000rpm.
• 4) Add to the pellet µl RLT buffer 600. Mix by pipetting.
• 5) Pipette the lysate directly into a column of the RNeasy mini kit, centrifuge for 2 min at maximum speed.
• 6) Add 1 volume of 70% ethanol to the homogenized lysate and mix well by pipetting. Do not centrifuge.
• 7) Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently and centrifuge for 15 s in ≥8000 xg (≥10,000rpm).
• 8) Add 500 µL of RPE buffer to the RNeasy spin column. Close the lid gently and centrifuge for 15 s in ≥8000 xg (≥10,000 rpm).
• 9) Add 500 µL of RPE buffer to the RNeasy spin column. Close the lid gently and centrifuge for 2 minutes at ≥8000 xg (≥10,000 rpm).
• 10) Place the RNeasy spin column in a new collection tube 1.5 ml (supplied). Add 30-50 µl RNAase free water directly onto the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥8000 xg (≥10,000 rpm) to elute the RNA.