Preparation of RNA from Strawberry and Other Recalcitrant Tissues

This method couples the qualities of CTAB with chloroform extraction to generate large amounts of RNA from strawberry or other tissues. This is a modification of the 'pine cone' method introduced by Chang et al., 1993 and amended for our use by Dawn Bies.

STOCK SOLUTIONS: 

10% CTAB
10% PVP (K 30) 
1 M TRIS (pH 8), some DEPC treated
0.5 M EDTA (pH 8), some DEPC treated
5 M NaCl
200 mg/ml Spermidine
Beta-mercapto ethanol
 Water, some DEPC treated 
10 M LiCl (DEPC treated) 
Chloroform/Isoamyl (24:1)    

 SSTE* 50 mls:

10 mls 5M NaCl=1M NaCl final
1.25 mls 20% SDS=O.5% SDS final
 500µl 1M Tris pH8=10mM final
100µl 0.5M EDTA=1mM final 

Extraction Buffer: 

2% CTAB
2% PVP (K 30)
100 mM Tris-Hcl
 25 mM EDTA  
2.0 M NaCl 
0.5 g/L spermidine2% BME  

 100 mls:

20 mls 10% CTAB
20 mls 10% PVP
10 mls 1M Tris
5 mls 0. 5M EDTA
40 mls 5M NaCl
250 ul 200 mg/ml spermidine
2 mls BME
2.75 mls Water

 

PROTOCOL

A ratio of 1g of tissue to 10 ml of extraction buffer is generally appropriate for most tissues. However, to maximize RNA yield all tissue types should be tested to determine the optimal tissue:buffer ratio. 

1. Warm extraction buffer to 65°C

2. Grind tissue in liquid Nitrogen and add to buffer, vortex,  place into 65 degree water bath 10 min (can go longer).

3. Add equal volumes of chloroform/isoamyl to each sample and homogenize with the polytron (or equivalent) for one minute at 80-90% max speed (PT-10/35 generator).

4. Separate phases at room temperature for 10 minutes @ 8,000 x g. 

5. Remove the supernatant to a new tube and add equal volumes of chloroform/isoamyl, vortex samples and separate phases again at R.T. for 10 minutes @ 8,000 x g. 

6. Add LiCl to a final concentration of 2.0M (1/5volume if using 12M LiCl or ¼ volume if using 10M LiCl)  and ppt. O.N. @ 4 degrees C in ice bucket. 

7. Pellet RNA by centrifugation at 4°C, 10,000 xg for 30 minutes.

8. Resuspend pellet in 500 µl SSTE.

9. Extract with chloroform:isoamyl once, centrifuge, and transfer supernatant to a clean 1.5 ml tube.

10. Add two volumes of 100% ice cold EtOH and ppt at -70°C for at least 30 min or -20C for at least two hours . 

11. Pellet RNA as before, wash in 76% ETOH, 0.3M NaOAC (DEPC treated).  

12. Dry pellet and resuspend in 10mM Tris, 2.5mM EDTA.

13. Check concentration using spectrophotometry.