#2 RNA Extraction from TISSUE using Trizol reagent (modified from
Gibco BRL/Invitrogen):



1.      Collect tissues in RNA later (O/N RT or 7 days 4C or frozen w/o
RNAlater at –80C)
2.      Aspirate RNAlater out
3.      Add Trizol (2mL to 0.1g tissue; for skin, use 3mL) to a round
bottom 15 ml polypropylene tube  ice.
4.      Transfer tissue (~0.1g or tail biopsy) to the Trizol tube on ice.
5.      Homogenize with Polytron on high speed (setting 6 or 7) for 45 sec
to 1.5 min or until well-homogenized.
6.      Incubate samples on ice, then at RT for 5 min to permit
dissociation of nucleoprotein complexes
7.      Split the Trizol homogenate into microcentrifuge tubes (1mL in each).
8.      Add 200ul Chloroform to each 1mL of homogenate,
9.      Cap the tubes well and shake vigorously by hand for 15 sec
10.     Incubate tubes at RT for 4 min
11.     Shake again each tube and centrifuge at 14,000 rpm 15 min 4C
-You will see 3 phases: red (phenol:chloroform), white (interfase,
where DNA will be), and aqueous colorless (where RNA will be).
-RNA remains exclusively in the aqueous phase.
12.     Transfer the aqueous phase to a new tube, label. Make sure not to
disturb the interphase (~400-550ul). Taking less is preferable to
taking any of the white stuff.
13.     Precipitate the RNA from the aqueous phase using Isopropanol
(500ul per 1mL of Trizol used earlier). Mix well by inverting several
times
14.     Incubate at RT 10 min or 1 hour on ice to precipitate
15.     Centrifuge samples at 14,000 rpm for 10 min at 4C
16.     Remove the supernatant (decant) and make sure the pellet is ok
17.     Wash RNA pellet with 80% cold (-10C) Ethanol. At least 1mL per 1mL
of trizol used earlier.
18.     Spin 10,000rpm 10 min 4C
19.     Remove supernatant (decant). Briefly dry the pellet (air-dry 10-15min)
20.     Dissolve RNA in 50ul depc-H2O. Can place at 55C for 10-15 min to
help. Can store samples at –80C or measure A260 (1:100 dilution; 2ul
sample + 198ul water).