(June 2006 Kathryn Brown)
Prior to starting the extractions, add 500µl Buffer RLT to 2mL screw top tubes half full of beads (they can soak overnight). Vortex and centrifuge to remove trapped air. Wipe surfaces down with RNase Away.
For cells collected on filters and frozen at –80° C use:
- determine the number of samples or extractions.
- Wash samples with Buffer RLT. For each sample, 1.2mL of Buffer RLT is needed with fresh addition of B-mercaptoethanol (stable for one month, 10µl B-mercaptoethanol per 1mL of buffer RLT). Tip: Keep tubes on ice. Keep pipette tip used to wash filter paper for transferring to 2mL screw top tubes
- Obtain pre-soaked beads in screw top tubes; add 1.2mL of sample to tubes. Bead beat 2 minutes with Mini-bead beater
- Centrifuge 3-4 minutes at max speed. Transfer ‘supe’ (discard pellet, the insoluble material) to the old labeled 15 mL conical tube (cleaned with a little 70% EtOH, ~0.4mL).
- Add an equal volume of 70% EtOH (made with 100% EtOH and DEPC water) to the cleared lysate and mix well by pipetting. Tip: Keep the same pipette tip used to transfer supe in the 15mL conical tube for applying the sample to the RNeasy mini spin columns
- Apply 700µl of sample at a time to labeled RNeasy mini spin column (in a 2ml collection tube). Centrifuge each time for 15 seconds.
- Repeat until the entire sample has passed through the column.
- Pipette 700µl of buffer RW1 onto the column, centrifuge 15 seconds.
- Discard flow through and collection tube
- Transfer RNeasy column to new collection tube. Pipette 500µl of Buffer RPE (ethanol added) onto the column and centrifuge 15 seconds. Discard flow through
- Pipette 500µl of RPE onto column again, and centrifuge for 2 minutes at max speed. Discard flow through and repeat empty.
- Transfer RNeasy column to a new labeled 1.5ml RNase free microcentrifuge tube. Pipette 40µl of RNase free water directly onto the RNeasy membrane. Centrifuge for 1 minute at 10,000 rpm to elute RNA.
- Store at –80oC.
Determine concentration and Purity: Spec and KC Junior
Reference: Brown, K.L., K.I. Twing, and D.L. Robertson. 2007.