Kafatos:totalRNA extraction from adult mosquitoes



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.