I. RNA extraction
Use can use your favorite method to extract RNA. Make sure that it is not degraded using
an agarose gel and quantify using a Nanodrop. We have used the following
1. Grind samples in a mortar and pestle with sand and liquid nitrogen for at least 10
min.
2. Transfer the powder to a Teflon tube. Add 10-20 ml/gfw of extraction buffer. Mix
vigorously for at least 15 min at RT
3. Extract for some minutes in 1 volume of chloroform / isoamyl alcohol (24:1) then
centrifuge 15 minutes 10,000g RT
4. Transfer the aqueous (top) phase to a small glass pot (yogurt pot) and precipitate
polysaccharides by adding 0.2-0.3 volumes of 100% ethanol drop by drop under
mild agitation
5. Extract for some minutes in 1 volume of chloroform / isoamyl alcohol (24:1) then
centrifuge 15 minutes 10,000g RT. Recover the aqueous phase.
6. Precipitate the RNA O/N at -20°C with ¼ volume of LiCl 12N and 1% v/v mercaptoethanol (mix vigorously)
7. Centrifuge 1 h at 12,000g 4°C
8. Discard the supernatant and invert the tube onto absorbing paper. (Note: The
supernatant contains DNA that can be precipitated and purified)
9. Use 100 µl Ambion Turbo DNAse buffer 1X to dissolve pellet. Add 10 U of
Turbo DNAse and incubate 30 min at 37°C. Add 200 µl of nuclease-freewater.
10. Resuspend on TE (1.5ml) (or in water) in eppendorf tubes
11. Extract with 1:1 phenol: chloroform (Note: Use phenol for RNA, pH 4.3)
12. Spin at 13,000 rpm, for 20 min
13. Recover the aqueous phase and precipitate the RNA with 1/10 volume of 3M
sodium acetate pH 5.5 and 2.5 volumes of ethanol
14. RNA can be stored at -20°C and recovered by centrifugation
Extraction buffer : 100mM Tris-HCl pH 7.5
1.5M NaCl
2% CTAB
50mM EDTA pH8
50mM DTT (ajouter extemporanément au tampon)
TE : Tris-HCl pH 8 10mM
EDTA 1mM