Protocol R.2
RNA Preparation from Cultured Cells or Tissue Samples
This protocol has been used to isolate RNA from relatively small tissue samples. The RNA is clean enough for RNase protection, cDNA synthesis, and RT-PCR analysis.
Solutions
Lysis Solution
4M GuSCN 250 g guanidine thiocyanate
25mM Na citrate 7.0 17.6 ml 0.75M Na citrate 7.0
0.5% Sarkosyl 26.4 ml 10% Sarkosyl
add 293 ml Q
before use, add 72ml bME
Water Saturate Phenol
thaw 500 ml phenol
add 0.5g hydroxyquinolin
add 500 ml Q
mix and allow phases to separate at room temperature
repeat 2 times and store at 4°C
3M NaOAc 5.2
24.6 g NaOAc (anhydrous)
pH to 5.2 with acetic acid
up to 100 ml Q
Procedure
• Pellet 10 ml cells (1x106 cells per ml) by spinning 1.2K for 10 minutes. Or, if using tissue samples, dissect the tissue in 1X PBS.
• Wash with 5 ml PBS.
• Add 500 ml Lysis Solution to cell pellet (this can be frozen or processed immediately).
• Add 1 ml Glycogen, 30 ml 3M NaOAc 5.2 and 500 ml water saturated phenol. Mix by gentle inversion and add 100 ml chloroform.
• Mix by inversion and hold on ice for 15 minutes.
• Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 ml isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
• Resuspend the pellet in 300 ml Lysis Solution and add 300 ml isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
• Resuspend the pellet in 50 ml DEPC treated TE and quantitate 5 ml. I get approximately 1-2 mg/ml.