In the course of our studies on expression of the sucrose synthetase genes, a simple and efficient procedure was developed for the isolation of high-integrity, translatable mRNA from several maize tissues. The protocol for large scale preparation of RNA from developing kernels is as follows:
1. Ears (20-22 days post-pollination) are harvested and frozen immediately in liquid N2 (this is done in the field). The frozen ears are stored at -90 C until needed.
2. 20 g of frozen kernels are thoroughly pulverized under liquid N2 with a mortar and pestel. Care must be taken to avoid thawing.
3. The frozen powder is added to a 250 ml
flask containing 60 ml of the following solution:
100 mM Tris-Hcl (pH 9.0)
200 mM NaCl
5 mM DTT
1% (w/v) sarcosyl
20 mM EDTA
The frozen tissue is then dispersed and thawed
by homogenization with a Polytron for 1 min. (Extensive foaming will
occur, but this is not harmful.)
4. The suspension is immediately transferred to 50 ml Oak Ridge centrifuge tubes and centrifuged at 10,000 xg for 5 min.
5. The supernatant is transferred to clean Oak Ridge tubes and immediately extracted with an equal volume of 50:50:1 phenol:chloroform:isoamyl alcohol and then centrifuged at 10,000 xg for 5 min.
6. The lower phase is removed and discarded leaving the interface and upper layer. An equal volume of chloroform/isoamyl (50:1) is added, mixed, and centrifuged as above.
7. The aqueous layer (upper) is transferred to a fresh tube and extracted a second time with chloroform/isoamyl (50:1). The aqueous upper phase is saved.
8. The volume of the extract is determined and the solution is adjusted to 2 M LiCl with a 12 M LiCl stock solution, mixed and transferred to clean, sterile Corex centrifuge tubes. These are allowed to stand overnight at 4 C.
9. The RNA is recovered by centrifugation at 10,000 xg for 10 min. The RNA pellets are thoroughly resuspended in 3 ml of 2 M LiCl and centrifuged saving the pellet. This is done twice.
10. The RNA is dissolved in 2 ml of 10 mM Tris-HCl (pH 7.5), 2 mM EDTA and centrifuged at 10,000 xg for 10 min to remove any insoluble material.
11. A 1/10 volume of 3 M Na Acetate (pH 5.2) is added followed by 2.5 volumes of ethanol. The mixture is incubated overnight at -20 C or for 1-2 h at -90 C then centrifuged at 10,000 xg for 10 min. The pellet is rinsed with 70% ethanol then resuspended in 10 mM Tris-Hcl (pH 7.5), 2 mM EDTA.
Notes: Several precautions should be taken to avoid RNase. All solutions should be autoclaved and kept sterile. Glassware should be thoroughly washed and baked at 220 C overnight before each use. Gloves should be worn at all times.
The resulting total cellular RNA is highly intact as judged by Northern blots probed with Sh1 and Ss2 clones. PolyA-RNA prepared from this material by oligo-dT cellulose chromatography produced abundant high molecular weight translation products (>90 kD) in vitro in a rabbit reticulocyte lysate system. The yield from 20 g of kernel tissue ranged from 10-12 mg of RNA. The A260/A280 of the RNA ranged from 2.0-2.2.
A modification of this procedure has been adapted to small sample sizes (less than 1 g) and used with other maize tissues. Frozen powdered tissue is added directly to a centrifuge tube and suspended in approximately 3 volumes of extraction buffer by vortexing. This suspension is immediately extracted with phenol/chloroform without prior centrifugation. The remainder of the procedure is as described above. Multiple samples are readily processed simultaneously with this protocol. We have obtained good results using this method with root and shoot tissues from seedlings.
We have not rigorously compared this procedure to others that are in use, however, the RNA we obtain compares very favorably in quality with that described in the literature. We have found this protocol to be straight-forward and effective with a wide range of sample sizes.
D.R. McCarty
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