Peter Hauser (last update 20.11.2002)

Kidney wedge biopsies were obtained right before implantation. A small part (about 12µg) of the wedge biopsy was put into an Eppendorf fube containing 500 µl RNAlater™ (Ambion, Austin, TX). Samples where stored at +4°C over night and kept at -80°C for long-time storage.

Procedures

• Put biopsy into 1 ml TRIzol®, grind tissue till a homogeneous mass emerges
• If frozen tissue is used; thaw on ice and remove RNAlater® with pipette
• Transfer grinded biopsy to an Eppendorf tube and add 200 µl chloroform
• Incubate in orbital shaker for 3 minutes
• Centrifuge (20 minutes, 10000 g, 4°C)
• Transfer the aqueous (upper) phase to a new Eppendorf tube
• Precipitate RNA by adding an equal amount (~550 µl) of isopropanol
• Incubate 10 minutes at room temperature
• Centrifuge precipitation mix (20 minutes, 10000 g, 4°C)
• Wash pellet in 1ml 70% ethanol
• Centrifuge again (10 minutes, 10000 g, 4°C)
• Air dry pellet at room temperature for 15 minutes
• Resuspend in 10µl nuclease free H2O

Materials: