Total-RNA-From-Rabbit-Spleen

Materials

1. Plasticware: 50-ml conical tubes (e.g., Falcon tubes; BD Biosciences, San Jose, CA); RNase-free 50-ml centrifuge tubes (e.g., Nalgene 3139 Oak Ridge centrifuge tubes; Thermo Fisher Scientific, Waltham, MA); RNase-free 1.5-ml microcentrifuge tubes (e.g., Biopur tubes; Eppendorf, Westbury, NY);

RNase-free filter pipette tips (e.g., ART; Molecular BioProducts, San Diego, CA).

2. Spleen and bone marrow from one femur of an immunized rabbit, each in 10 ml TRI reagent (Molecular Research Center,
Cincinnati, OH; store at 4 oC) in 50-ml conical tubes stored on dry ice or at -80 oC (see Note 1).

3. BCP: 1-bromo-3-chloro-propane (Molecular Research Center). Store at room temperature.

4. Isopropanol (Sigma-Aldrich, St. Louis, MO). Store at room temperature.

5. Freshly prepare 70% (v/v) ethanol (Sigma-Aldrich) in RNasefree water (Ambion, Austin, TX) and keep at roomtemperature.

6. RNA storage solution: RNase-free 1 mM sodium citrate (pH 6.4) (Ambion). Store at -20oC.

7. RNase-free 3 M sodium acetate (pH 5.2) (Ambion). Store at room temperature.

Method

1. For total RNA preparation, process rabbit spleen and bone marrow in 10 ml TRI reagent in 50-ml conical tubes in parallel (see Note 5). Typically, tissues from two rabbits are processed as four samples in parallel. Start by thawing the samples for 15 min at 37 oC in a water bath, blend with a homogenizer, for example a PowerGen 125 (Thermo Fisher Scientific) at 50% output for 1 min, and incubate for 5 min at room temperature.

2. Add TRI reagent to a final volume of 30 ml and centrifuge at 2500 g for 10 min at 4oC.

3. Transfer the supernatant to an RNase-free 50-ml centrifuge tube (discard the pellet), add 3 ml BCP, vortex for 15 s, incubate for 15 min at room temperature, and centrifuge at 17,500 g for 15 min at 4oC.

4. Transfer the upper colorless aqueous phase to a clean RNasefree 50-ml centrifuge tube (discard the lower red organic phase), add 15 ml isopropanol, vortex for 15 s, incubate for 10 min at room temperature, and centrifuge at 17,500 g for 10 min at 4 oC.

5. Carefully decant and discard the supernatant without disturbing the white pellet, add 30 ml 70% (v/v) ethanol, and centrifuge at 17,500 g for 10 min at 4 oC.

6. Carefully decant and discard the supernatant without disturbing the white pellet, air-dry in dust-free conditions for 10 min at room temperature, dissolve in 500 ml RNA storage solution, and transfer to an RNase-free 1.5-ml microcentrifuge tube.

7. Immediately remove a 2-ml aliquot and store the remaining sample on dry ice. Add 498 ml RNase-free water to the 2-ml aliquot and measure the absorbance at 260 nm and 280 nm in a UV photometer. Use the absorbance at 260 nm to calculate the total RNA concentration based on the assumption that 40 mg/ml RNA gives an absorbance of 1 (see Note 6).

8. Store total RNA at -80 oC. For long-term storage, add 0.1 volume RNase-free 3 M sodium acetate (pH 5.2) and 2.2 volumes ethanol, vortex, and store at -80 oC.