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RNA extraction using RNeasy kit

  1. Wipe down all surfaces with RNeasy inhibitor solution
  2. For each sample, label:
    • one QiaShredder (in collection tube)
    • one RNeasy column (in collecting tube)
    • one 2 mL collection tube
    • one 1.5 mL Eppendorf tube (sterile)
  3. Allow samples (in RTL buffer) to thaw at room temperature
  4. Pipette 200 μL of lysed samples into QiaShredder. Centrifuge for 2 minutes at 13,000 rpm.
  5. Add 200 μL of 70% ethanol to the transferred lysate in the collection tube; mix well by pipetting.
  6. Transfer RLT/EtOH mixture into the RNeasy column. Spin for 15 seconds at 13,000 rpm. Discard flow-through.
  7. Add 700 μL of buffer RW1 to the column. Centrifuge for 15 seconds at 10,000 rpm.
  8. Transfer RNeasy column to a new 2 mL collection tube. Add 500 μL of buffer RPE to the column and centrifuge for 15 seconds at 10,000 rpm.
  9. Discard the flow-through and repeat step 8 (use the same collection tube).
  10. Discard the flow-through. Dry the column by centrifuging at 13,000 rpm for 2 minutes.
  11. Elute RNA: place the column in the 1.5 mL Eppendorf tube and add 35 μL of RNase free water directly onto the filter of the RNeasy column. Leave for 1.5 minutes at room temperature. Then spin the 1.5 minute at 13,200 rpm.
  12. Estimate [RNA] using Nanodrop and store samples at -80 ̊C.