Method for extracting total RNA from fungal hyphae

Reagents:

RNA extraction buffer (CTAB): 2% CTAB (W/V), 2% polyvinylpyrrolidone PVP (W/V), 100 mM Tris-HCl (pH 8.0, DEPC treated water configuration), 25 mM EDTA, 0.5 g/L spermidine Spermidine, 2.0 M NaCl, 2% mercaptoethanol (V/V, added before use). Since Tris-HCl reacts with DEPC under high temperature sterilization conditions, it can be prepared directly with DEPC-treated water when using RNA extraction buffer.

SSTE: 1 M NaCl, 0.5% SDS (W/V), 10 mM Tris-HCl pH 8.0, 1.0 mM EDTA

10M LiCl was directly diluted with distilled water to 10M LiCl, and 1 liter of DEPC was added overnight, and then sterilized by high temperature.

3M NaAc

Chloroform: isoamyl alcohol (24:1)

Phenol (pH 4.5): chloroform: isoamyl alcohol (25:24:1)

DEPC treatment water treatment of distilled water with 1 ‰ DEPC overnight, high temperature sterilization

Absolute ethanol

70% alcohol

method

1. Before the start of the experiment, the RNA extract was preheated in a 65 ° C water bath, and ME (mercaptoethanol) (10 mL plus 80 ul, 300 ul in 300 mL) was added to the centrifuge tube.

2. Take about 0.8g of mycelium (the hyphae obtained by liquid culture can be vacuum filtered; solid culture is better), quickly grind into fine powder in liquid nitrogen, put into a 50 mL centrifuge tube, press 1g Add 8 mL of material to the pre-warmed RNA extract and mix by inversion.

3. 65 ° C water bath 3-10 min, mixing 2-3 times during the period

4. Add an equal volume of phenol (note acid phenol pH 4.5): chloroform: isoamyl alcohol (25:24:1) extraction (10,000 rpm, 4 ° C, 5 min)

5. Take the supernatant and extract an equal volume of chloroform: isoamyl alcohol (24:1) (10,000 rpm, 4 ° C, 5 min)

6. Add 1/4V volume 10M LiCl solution and place at 4 °C for more than 6hr (or overnight)

7. Centrifuge at 10,000 rpm for 20 min at 4 °C

8. Discard the supernatant and dissolve the pellet with 500 ul SSTE

9. Phenol: chloroform: isoamyl alcohol (25:24:1) was extracted twice, and chloroform: isoamyl alcohol (24:1) was extracted once (10,000 rpm, 4 ° C, 5 min)

10. Add 2V volume of absolute ethanol and precipitate in the refrigerator at -70 °C for more than 30 min.

11. 12,000 rpm, centrifuged at 4 ° C for 20 min

12. Discard the supernatant. The precipitate is rinsed once with 70% alcohol and dried.

13. Add 200 ul of DEPC to dissolve the water

14. Detect the quality of RNA by non-denaturing agarose gel electrophoresis and UV spectrophotometry. (In the extraction process, if the protein content or other impurities are more, the number of extractions can be increased.)
Note: Please extract the RNA at low temperature as much as possible. If the conditions are not allowed, operate at room temperature as short as possible.