Protocol 12 Hybrid protocol for the isolation of total RNA

Follow the phenol/chloroform method (Tri-Reagent, Trizol, RNAsol, etc) with the following modifications:

Step 1: Homogenize tissue samples

1) Homogenize tissue samples in a phenol-based reagent in the amounts indicated:

The amount of reagent required may be greater for certain tissues; e.g., brain. If you don't get a clear aqueous phase, add another ml of homoge-nization reagent, vortex and spin. Repeat if necessary.

Tissue (mg) Phenol Reagent (ml) 100 1 ml 500 5 ml

1000 10 ml

2) Add tissue to the phenol reagent. Use a power homogenizer to homogenize tissue for 1 min or until there are no tissue chunks (settings should be determined and optimized for the specific instrument). If tissue culture cells are used, the phenol-based reagent can be added directly to the plates after the media is removed. Cells removed by scraping.

3) Separate insoluble material from the homogenate by centrifugation at 12,000 x g for 10 min at 2-4°C. The supernatant contains the RNA while the pellet contains extracellular membranes. If working with adipose tissue, remove the supernatant from under fat that collects as a top layer.

4) Transfer the cleared supernatant to a fresh tube being careful not to carryover any of the interface. Add 0.2 ml of chloroform for every 1.0 ml of phenol reagent.

Step 2: Separate phases

1) Securely cap the tube from 4 above. Shake the tube vigorously for 2 min (end to end).

2) Incubate at room temperature for 2 min.

3) Centrifuge at 12,000 x g for 15 min, 2-4°C. The resulting sample should show 3 phases:

1-Lower red (Tri-Reagent) phenol-chloroform phase

2-White interphase

3-Upper colorless aqueous phase

The RNA remains exclusively in the upper aqueous phase. If the aqueous phase appears cloudy, you may perform the chloroform extraction step up to 3 times.

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