RNA Extraction from Infected Maize Kernels

Materials and Supplies

Mortar and pestle

1.5ml tubes

DEPC water

Tris EDTA buffer (ACROS Organics: DNase, RNase, and Protease free, ready to use,

pH 8.0, Cat # AC32734-5000)

5:1 Acid Phenol: Chloroform (pH 4.5, Cat #9720)

75% ethanol

Saturated Phenol (pH 6.6, Cat# 9712)

RNase Away

Isopropanol

High Salt (DEPC treat): 0.8M Sodium Citrate, 1.2M NaCl

 

Procedure

 

  1. Clean surfaces with RNase Awayรข
  2. Take 8 kernels. Grind thoroughly until it is at the consistency of powder and store at -80oC
  3. Pipette 0.75mL of phenol into 15ml tubes
  4. Add 1 scoop of grinded tissue sample to the 0.75mL phenol. Homogenize 4min
  5. Add 0.75mL tris buffer to the 0.75mL phenol. Vortex 2min.
  6. Centrifuge at 14K RPM, 4oC, 10min.
  7. Without disturbing the interface, transfer the aqueous layer (top layer) to another 1.5ml tube containing 0.75ml of Phenol: Chloroform. Vortex 2min and spin at 14K RPM, 4oC, 10min.
  8. Without disturbing the interface, transfer the aqueous layer (top layer) for a 2nd time to another 1.5ml tube containing 0.75ml of Chloroform. Vortex 2min and spin at 14K RPM, 4oC, 10min.
  9. Carefully remove the aqueous layer to a 1.5ml tube. Add 250ul isopropanol (mix) and 250ul high salt (mix). Incubate at -20oC for 1 hour.
  10. Spin at 14K RPM, 4oC, 30 min.
  11. Decant supernatant, and wash with 75% ethanol.
  12. Spin at 6K RPM, 4oC, 6 min. Briefly dry pellet -6 minutes (not too long, or pellet will be extremely difficult to re-suspended).
  13. Re-suspend in 60ul DEPC water with 1ul RNasin

 

RNA can be further purified using the Qiagen RNeasy cleanup protocol