Table 1

Overview on the tested stool protocols

Protocol 1Protocol 2Protocol 3Protocol 4Protocol 5Protocol 6Protocol 7Protocol 8
50 mg stool50 mg stool50 mg stool50 mg stool50 mg stool50 mg stool50 mg stool50 mg stool
150 µL PBS200 µL isopropanol200 µL Methanol/ acetonitril/ H2O * (2/2/1)200 µL Methanol/ acetonitril (1/1)150 µl 85% Ethanol/15% 20 mM phosphate buffer200 µL 75% Ethanol225 µL 100% Methanol150 µL 100% methanol
Vortex 2 min
Freeze in liquid N2 (1 min); thaw
5 min sonification on ice
500 µL MTBE750 µL MTBE
Shake 1 hour at RTShake 1 hour at 4°C
188 µL H2O * + 0.1% ammonium acetate
Vortex 2 min, incubate 5 min at RT
Centrifugation at full speed (5 min)Centrifugation at full speed (10 min)Centrifugation at full speed (5 min)
SupernatantUpper supernatantSupernatant
150 µL 20% Ethanol/80% 20 mM phosphate buffer125 µL H2O *~2000 µL 100% methanol150 µL 20% methanol
Vortex 2 minVortex 1 min
Incubate 10 min at RTIncubate 1 hour at −20°C
Centrifugation at full speed at 4°C (15 min) + supernatant in extra tube
Freeze in liquid nitrogenFreeze in liquid nitrogenFreeze in liquid nitrogenFreeze in liquid nitrogenFreeze in liquid nitrogenDry complete in SpeedVacDry complete in SpeedVacFreeze in liquid nitrogen
  • *DNAse-free and RNAse-free water.

  • MTBE, methyl tert-butyl ether; PBS, phosphate-buffered saline; RT, room temperature.