RNA isolation with Trizol reagent


Please take precautions when working with Trizol (contains phenol): gloves, hood
  • collect embryos in 2ml tube (1 mg wet weight corresponds to about 100 embryos)
  • add 300 µl Trizol, you may add less for easier homogenisation and add the remainder afterwards
  • homogenize embryos with micropestle
  • spin in microfuge 12000 rpm, 10min, 4°C
  • transfer supernatant (aqueous phase) into a 1,5ml tube
  • incubate for 5 min at room temperature
  • add 60 µl chloroform
  • mix for 15 s
  • incubate for 2 to 3 min RT
  • spin 12000rpm, 15min, 4°C
  • the clear upper phase contains the RNA
  • transfer only upper phase into new tube
  • precipitate RNA: 75 µl 2-propanol and 75 µl 0,8M NaCitrat + 1,2 M NaCl (per 300µl Trizol)
  • incubate for 10min at room temperature
  •  spin 10 min, 4°C
  • wash pellet with 400 µl 70% EtOH
  • spin 5min, 4°C
  • repeat wash and centrifugation
  • dry pellet in speed vac for about 1min
  • dissolve in 10-20 µl DEPC H2O
  • meassure absorption at OD260
  • freeze RNA samples in liquid nitrogen, store in -80°C freezer

    • Typical yield:    50 to 150 ng total RNA per embryo