• Wash cells with PBS
• Add 300 µl (3.5cm well) hypotonic lysis buffer to cells
• Shake plates gently to make sure buffer washes over all cells
• Incubate about 1 min.
• Transfer 200 µl (of the 300 total) lysate to 1 ml of Trizol.
• Prepare RNA according to manufacturer:
• Leave Trizol mixture at RT, 5 min.
• Add 200 µl Chloroform.
• Mix by shaking for 30 sec or more.
• Leave at RT, 10 min.
• Spin 14,000 rpm, 4C, 15 min.
• Transfer supernatant to new tube. Avoid interphase!!
• Add 0.7 volumes of isopropanol. Mix and leave at RT, 10 min.
• Spin 10,000 rpm (not faster!), 4C, 30 min.
• Remove supernatant carefully.
• Wash with 70% EtOH.
• Dry.
• Dissolve in 20 µl formamide load buffer.

Buffers:

• Hypotonic gentle lysis buffer:

  10 mM Tris-HCl pH 7.5
  10 mM NaCl
  10 mM EDTA
  0.5% Triton-X100

• Formamide load buffer:

  Deionized Formamide
  5 mM EDTA
  0.1% Bromophenol blue
  0.1% Xylene Cyanol