Reagents:
3 mL extraction buffer (150 mM Tris base (hydroxymethyl-hydrochloride)
4% (w/v) SDS (100 mM EDTA, adjusted to pH 7.5 with saturated boric acid)
30 mL b-mercaptoethanol (2%, v/v)
3% (w/v) PVP-40
66 mL 5 mM potassium acetate
150 mL absolute etanol
850 mL chloroformisoamylalcohol (49:1, v/v)
850 mL phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v)
12 M LiCl
70% ethanol (500 mL each)
10 mL DEPC-treated water
Small-scale Valderrama-Cháirez et al. protocol (VC protocol)
Fresh in vitro banana leaves (0.5 g) were frozen with liquid N2 and powdered for 5 min using a mortar and a
pestle previously chilled to -20°C with liquid N2 added continuously. Small portions of powdered sample were added
to a vial containing 3 mL extraction buffer (150 mM Tris base (hydroxymethyl-hydrochloride), 2% (w/v) SDS, 100 mM
EDTA, adjusted to pH 7.5 with saturated boric acid), and 30 mL b-mercaptoethanol (1%, v/v) added just before use.
This suspension was quickly mixed using a cut tip to avoid RNA damage, transferred to Eppendorf tubes (750 mL per
tube), precipitated with 66 mL 5 mM potassium acetate, and 150 mL absolute ethanol, and vortexed for 1 min. Next,
850 mL chloroform-isoamyl alcohol (49:1, v/v) was added, and the suspension vortexed for 10 s and centrifuged at
16,000 g for 20 min at room temperature. The supernatants were recovered and transferred to new tubes, 850 mL
phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v) was added per tube, and the tubes were vortexed for 10 s and
centrifuged at 16,000 g for 15 min at room temperature. The supernatants were recovered and transferred to new
tubes, along with 850 mL chloroform-isoamyl alcohol. The tubes were vortexed for 10 s and centrifuged at 16,000 g
for 15 min at 4°C. The supernatants were recovered, 12 M LiCl was added to a final concentration of 3 M, and the
tubes were gently mixed by inversion and left to stand overnight at -20°C. The samples were then centrifuged at
16,000 g for 20 min at 4°C, and the pellets were washed twice with 70% ethanol (500 mL each) and centrifuged again
at 16,000 g for 10 min at 4°C. The pellets were dried at room temperature and resuspended in 10 mL DEPC-treated
sterile distilled water. The suspensions were pooled in a new tube, and RNA quantified in a UV-light
spectrophotometer. RNA was stored at -80°C until use.
Modified small-scale Valderrama-Cháirez et al. protocol (MVC protocol)
This was carried out as described above for the VC protocol, but with some modifications: 4% (w/v) SDS and
2% (v/v) b-mercaptoethanol, and the addition of 3% (w/v) PVP-40 in the extraction buffer (3 mL), where the last two
reagents were added just before use. An additional step was added, the incubation of the homogenate (material
powdered with modified extraction buffer) at 65°C for 10 min. After allowing the samples to return to room
temperature, potassium acetate and absolute ethanol were then added.
Optimized procedure
1. Weigh 4g of tissue and freeze with liquid nitrogen. Avoid thawing the tissue to keep hydrolytic enzymes
(ribonucleases) inactive.
2. Grind the tissue to a fine powder in a mortar.
3. Transfer the powdered tissue to a new polypropylene conical tube. Quickly add 10 mL of extraction
buffer. Vortex for 1 min.
4. Precipitate the homogenate by adding 1.1 mL of 5 M potassium acetate and 2.5 mL of absolute ethanol.
Vortex for 1 min. At this stage, the suspension should appear flocculent.
5. Extract with 15 mL of chloroform-isoamylalcohol. Vortex and centrifuge at 27,200 g for 15 min. There
should be a colored nonaqueous bottom layer, a thick intermediate layer with cellular debris, and an
aqueous top layer. If not correctly centrifuged, some of the cellular debris will remain in the aqueous
phase.
6. Remove the top layer with a micropipette. Extract again with 15 mL of phenol-chlorofom-isoamylalcohol.
Vortex, and centrifuge at 27,200 g for 15 min. When transferring the aqueous layer, the thin white
interface must not be re-moved.
7. Recover the top layer again. Extract once more with 15 mL of chlorofom-isoamylalcohol. Vortex and
centrifuge at 27,200 g for 15 min. All traces of the white interface should be gone.
8. Add LiCl to make a final concentration of 3 M. Mix gently by inversion. Precipitate overnight at -20oC. If
the RNA concentration is adequate, the solution will appear cloudy. If not, it will appear translucent.
9. Centrifuge at 31,000 gat 4°C for 45 min to recover a clear-to-white RNA pellet. Continue centrifugation for
another 45 min to recover the DNA.
10. Wash the RNA pellet with 1.5 mL of chilled 70% ethanol. Eliminate the alcohol excess with a micropipette
and air dry. Transfer the pellet to a new micro centrifuge tube and work with small volumes.
11. Resuspend the pellet in 200 µL of DEPC-treated distilled water. Quantitate spectrophotometrically.