Table 1.

Protocol developed for high quality RNA isolation from high fat, subcutaneous adipose tissues.

Homogenize 1 g adipose tissue in 5 mL of cold TRIzol solution for 30s. Centrifuge at 2600 x g for 30 min at 2 °C. Remove excess fat layer. Transfer cleared homogenate to clean tube. Incubate for 5 min. at 22 °C. Add 0.2 ml of chloroform per ml of TRIzol reagent. Cap tubes and shake vigorously for 15s. Incubate for 2–3 min. at 22 °C. Centrifuge at 2600 x g for 30 min. Transfer clear aqueous layer to clean tube. Add 0.57x the amount of aqueous layer in isopropanol to the tube and mix. Load onto PureYield™ RNA Midiprep System clearing column (Promega). Elute under vacuum. Wash with 20 mL RNA wash solution under vacuum. Place column in clean 50 mL tube and 1 ml of RNase-free water to top of column. Centrifuge at 2000 x g for 3 min. Quantify RNA. Approximate yield of 35 ug/g of subcutaneous bovine adipose tissue with 260/280 ratios of >1.8 using this method.