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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
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