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Arabidopsis RNA Prep

The following is an excellent method to obtain high-quality RNA from recalcitrant tissues, working especially well on etiolated Arabidopsis. Although this preparation is time consuming (approximately 6 hours over three days), the quantity and quality of yield justifies the investment.

From Bies and Folta, 2004, Analytical Biochemistry

RNA may be prepared as described as follows using either the TIPS (Spectrum Chemical, New Brunswick, NJ) or DIPS (Cactus Biotech, Hayward, CA) reagent from 0.5 g of fresh plant tissue. All steps were performed at or below 4 °C using RNAse-free reagents and glassware.

1. The tissue was frozen in liquid nitrogen and ground to a powder using a mortar and pestle.

2. The powder was transferred into several volumes of ice-cold grinding buffer (4% -aminosalicylic acid, 1% TIPS or DIPS, 50 mM Tris–HCl, pH 8.0, and 2% (v/v) -mercaptoethanol). Arabidopsis tissue was processed in 5 vol (w/v) of buffer. RNA preparations from other species, such as strawberry and tomato, were optimally extracted in larger volumes that must be experimentally determined for each species and tissue type.

3. An equivalent volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, and the mixture was homogenized by a Polytron (or equivalent) using a T10-35 generator at 80% maximum speed for 1 min.

4. The homogenate was centrifuged at 8000g for 10 min, and the clear supernatant was transferred to a clean, sterile, RNAse-free tube. Turbid supernatants were reextracted with chloroform:isoamyl alcohol (24:1) and then centrifuged at 8000g to generate a clear supernatant.

5. LiCl was added to 2.0 M to selectively precipitate RNA overnight at 4 °C, followed by centrifugation at 8000g for 20 min.

6. The supernatant was discarded, and the pellet was resuspended in a small volume (~500 l) of "resuspension buffer" (10 mM Tris–HCl, pH 8.0, 2.5 mM EDTA).

7, LiCl was added to 2.0 M, and RNA was again precipitated overnight, followed by recovery of the pellet through centrifugation at 12,000g for 20 min.

8. The pellet was washed once with 500 l 76% ethanol containing 0.2 M sodium acetate.

9. The pellet was air dried and resuspended in 50 l resuspension buffer prior to spectrophotometric and other downstream analyses.