Kafatos:totalRNA extraction from adult mosquitoes

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totalRNA extraction from adult mosquitoes

  1. Add 9 volumes TRIZOL to 1 volume of tissue (400μl of Trizol for 1-20 mosquitoes works fine)
  2. Homogenize
  3. Spin for 10 min at 12 krpm, 4˚C and transfer clear supernatant into a fresh tube.
  4. Incubate at RT for 5 min
  5. Add 0.2 ml CHCl3 per 1 ml Trizol. Shake vigorously by hand and incubate at RT for 5 min.
  6. Spin 15 min at 4˚C, 12 krpm. Transfer aqueous (upper) phase into a fresh tube.
  7. Add 0.5 ml Iso-propanol per 1ml Trizol, mix and incubate for 10 min @ RT
  8. Spin for 10 min @ 4˚C, 12 krpm.
Use these steps to clean up the sample for more sensitive reactions:
a. remove supernatant and resuspend in 300μl ddH2O
b. add 30μl 3M NaOAc pH 3
c. precipitate with 600μl abs. EtOH 15 min @ -80˚C
9. Remove supernatant and wash pellet with 1ml 75% EtOH per 1ml Trizol. Vortex.
10. spin for 5 min @ 4˚C, 8 krpm
11. Air- or speed-vac-dry pellet and dissolve RNA ~70μl H2O
try these tips from Antonio to increase final yield:
  step 7: use 0.8 volumes Iso-propanol per 1 volume Trizol, mix and precipitate 30 min @ -80˚C
  step 8: spin 30 min @ @ 4˚C, max krpm.
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