trizol_RNA_extraction.md

TRIzol RNA extraction

• Grind your samples 2 x 45sec at maximum frequency (30 Hertz) in TissueLyser (QIAGEN), use pre-cooled adapters

• Add 500 µL of TRIzol to the frozen samples, close tubes and mix vigorously until the sample is homogeneous

• Incubate the samples for 5 min at room temperature

• Briefly spin down the samples

• Add 100 µl of chloroform, close the tubes and shake tubes 15-20 sec vigorously by hand

• Incubate the samples for 3 min at room temperature

• Centrifuge the samples at 10.000 x g for 15 min at 4°C

• Transfer 150-300 µl of the clear, aqueous, upper phase to new tubes

• Add same volume of isopropyl alcohol, gently invert 8x, do not vortex

• Incubate the samples for 10 min at room temperature

• Centrifuge the samples at 10.000 x g for 10 min at 4°C

• Remove the supernatant

• Wash the pellet with 500 µl of 75% ethanol

• Centrifuge samples at 10.000 x g for 2 min at 4°C

• Remove the supernatant

• Dry the RNA pellet at least 10-30 min

• Add 60 µL of DEPC-water and incubate at 4°C over night

• Perform DNAse treatment (see ThermoFisher_DNAseI protocol)

• Store RNA at -80°C