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RNA Precipitation

Note: In general, overnight precipitation gives better yields for all of these protocols.

Isopropanol Precipitation

  1. Bring up volume to 135 µl and add 15 µl ammonium acetate solution (5M NH4OAc, 100mM EDTA).
  2. Extract 1X with phenol:chloroform.
  3. Extract 1X with chloroform.
  4. Precipitate RNA with 1 volume isopropanol for 30 minutes at -20°C.
  5. Spin down precipitate 15 minutes at 14,000 rpm at 4°C.
  6. Wash 1X with 70% ethanol/DEPC
  7. Resuspend in RNase-free water or formamide.

Lithium Chloride (LiCl) Precipitation

Notes:

  • LiCl precipitation does not efficiently precipitate RNA < 300 nts. Use isopropanol precipitation instead
  • Efficient LiCl precipitation requires >0.2 µg/µl RNA
  1. Bring volume up to 50 µl with RNase-free water.
  2. Add 1/2 volume (25 µl) LiCl solution.
  3. Incubate >1 hour at -20°C.
  4. Spin down 15 minutes at 14,000 rpm to pellet RNA.
  5. Wash pellet with 70% ethanol/DEPC.
  6. Resuspend RNA in DEPC water (0.1%).

Precipitation from Formamide Resuspension

Note: Suspension in formamide protects the RNA from degradation by RNases.

  1. RNA can be precipitated from formamide by adding 4 volumes of 100% ethanol at -20°C for 30 minutes to 1 hour.
  2. Centrifuging at 14,000 rpm for 10 minutes at 4°C.
  3. Wash pellet with 70% ethanol/DEPC.
  4. Resuspend RNA in DEPC water (0.1%).

© 2014 The University of Texas MD Anderson Cancer Center