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Total RNA Isolation Protocol 1,2,3

  1. Sample Preparation:
    1. To extract RNA from washed and pelleted cultured cells, add 200 l 4 M Guanidinium Isothiocyanate Solution (4M Guanidinium Isothiocyanate, 25 mM Sodium Citrate, pH 7.0, 1 M b-Mercaptoethanol) to 0.5 x 104 cells - 1 x 106 cells.
    2. To directly extract RNA from cultured cells growing in monolayer, add 200 l 4 M Guanidinium Isothiocyanate Solution directly to each well of a 6, 12 or 24 well plate. Add 100 l of the 4 M Guanidinium Isothiocyanate Solution directly to each well of a 48 or 96 well plate.
  2. Homogenize monolayer cells by pipetting the mixture up and down several times, taking care to "wash" cell material free from the culture dish, tube or well in the process. Homogenize washed, pelleted cells by pipetting the mixture up and down until the pellet is fully suspended. Use a small bore pipet to collect the cell homogenate.
  3. Transfer all of the homogenate to a pre-spun (12,00016,000 x g for 12 minutes) PLG 2 ml Heavy tube.
  4. Add 20 l (10 l per sample for 48 or 96 well plates) 2.0 M Sodium Acetate, pH 4.0 to the sample, cap the PLG tube and mix briefly.
  5. Add 200 l (100 l per sample for 48 or 96 well plates) water-saturated Phenol to the sample, cap the PLG tube, and mix thoroughly by repeated inversion. Do not vortex.
  6. Add 60 l (30 l per sample for 48 or 96 well plates) Chloroform: Isoamyl Alcohol (CI, 49:1) to the sample in the same PLG tube and mix thoroughly by repeated gentle inversion. Do not vortex.
  7. Incubate on ice for 10 minutes.
  8. Centrifuge at 12,00016,000 x g for 5 minutes in a microcentrifuge to separate the phases.
  9. Add 200 l (100 l per sample from 48 or 96 well plates) Phenol-Chloroform-Isoamyl Alcohol (PCI, 50:49:1) to the aqueous phase in the same PLG tube. Mix thoroughly by repeated gentle inversion. Do not vortex.
  10. Centrifuge at 12,00016,000 x g for 5 minutes to separate the phases.
  11. Collect resultant aqueous phase to an RNase-free microcentrifuge tube, add an equal volume of 100% Isopropanol, and mix by repeated inversion.
  12. Centrifuge at 12,00016,000 x g for 20 minutes.
  13. Discard resultant supernatant and wash pellet several times with 200 l 70% Ethanol, using 23 minutes at 12,00016,000 x g to re-pellet the sample.
    Note: Samples may be stored in the 70% Ethanol wash at this stage at 70C or colder for extended periods.
  14. Discard final wash and dry pellet at room temperature.
  15. Re-dissolve pellet in a suitable volume (510 l) of RNase-free water. Store the RNA solution at 70C.
    Note: Absorbance determinations should be performed in RNase-free TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0).15
References
  1. Chirgwin, J.M. et al. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 18:5294-5299.
  2. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guan idinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.
  3. Birnboim, H.C. (1988) Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis. Nucl. Acids Res. 16:1487-1497.
  4. Wilfinger, W.W. et al. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474-481.

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