RNALATER Protocol

By Grayson DuRaine

5/10/2005

STORAGE

  1. Suspend cells or tissue in approx. 10 volumes of RNAlater, do not freeze or use frozen tissue for this, it must be fresh.

  1. Place at 4C for overnight or up to 1 week, for archival storage transfer to -20C after overnight 4C storage.  To store in the -80C you must remove the RNAlater from the sample.

EXTRACTION

  1. Remove RNAlater from the sample, for cells centrifuge at ~3000g for 5 minutes and remove the supernatant, for tissues drain RNAlater.

  1. Homogenize/disrupt sample carefully in 1ml of QIAZOL lysis reagent. For cells a passage through a 20 to 22 gauge needle 6-7 times works well.  For tougher tissues homogenize in a rotor stator homogenizer, larger volumes of QIAZOL may be necessary adjust the protocol accordingly.

  1. Incubate at room temperature for 5 minutes.

  1. Add 200µl of Chloroform for 1ml of QIAZOL used and shake by hand for 15 seconds.

  1. Incubate at room temperature for 2-3 minutes.

  1. Centrifuge at 12000g for 15 minutes at 4C, solution will separate into an upper aqueous phase (RNA), a white interphase (DNA), and a red organic phase (PROTEIN, ETC,.).  

  1. Transfer the upper aqueous phase to a new 1.5ml tube.

  1. Add 1 volume (~600µl) 70% Ethanol and Vortex, DON’T centrifuge. Go to the next step quickly.

  1. Add 700µl of sample and any precipitate into an RNeasy spin column, centrifuge at >8000g for 15s at room temperature.

  1. Discard flow-through and Reuse 2ml collection tube.

  1. Repeat previous 2 steps for the remainder of the sample.  If the volume is extremely large consider splitting into two RNeasy spin columns.

  1. IF doing the on-column DNase digestion go to the next step. IF NOT dd 700µl of RW1 and centrifuge at >8000g for 15s at room temperature. Discard flow-through and 2ml collection tube. And go to step 19

  1. Add 350µl of RW1 and centrifuge at >8000g for 15s at room temperature and discard the flow-through.

  1. Add 10µl of DNase I stock solution to 70µl Buffer RDD . Mix by gently inverting the tube. DO NOT VORTEX! DNase is extremely sensitive to physical denaturation.

  1. Pipet the DNase I incubation mix 80µl directly onto the RNeasy membrane and allow to sit at room temperature for 15 minutes.

  1. Add 350µl of RW1 and centrifuge at >8000g for 15s at room temperature and discard the flow-through.

  1. Transfer to a new 2ml collection tube, and add 500µl RPE and centrifuge at >8000g for 15s at room temperature and discard the flow-through.

  1. Add 500µl RPE again and centrifuge at >8000g for 2 minutes at room temperature and discard the flow-through.

  1. Centrifuge dry at >8000g for 1 minutes at room temperature and discard flow-through and 2ml collection tube.

  1. Transfer RNeasy spin column to a new 1.5ml tube, and add 50µl of RNase free water to the membrane.  Centrifuge at >8000g for 1 minute at room temperature to elute the RNA.

  1. Repeat elution step with another 50µl of RNase free water.