P-MEXP-9260 - P-MEXP-9260

The frozen plant tissue was quickly ground to a fine powder using a mortar chilled in liquid nitrogen. The frozen powder was carefully transferred into 1.5 ml microcentrifuge tubes (~100 mg/tube) using small funnels and rubber spatulas also cooled with liquid nitrogen, . The frozen tissue was homogenized in 1 mL of TRIzol reagent (Invitrogen, 15596-026) then incubated at room temperature for at least 5 min with agitation. Chloroform (200 ul) was added to each sample and the tubes were vortexed or forcefully shaken by hand for 15 sec. After 2-3 min of incubation at room temperature, the samples were centrifuged for 15 min at 4°C at 12000xg. The aqueous layer was transferred to a new 1.5 mL tube and 500 ul of isopropanol was added. The mixture was gently inverted to mix and incubated for 10 min at room temperature before centrifuging at 12000xg, 4°C, for 10 min. The pellet was washed with 75% ethanol (in DEPC-H2O) and centrifuged for 5 min at 12000xg and 4°C. The pellet was air-dried for 5 min at room temperature then resuspended for purification. The total RNA was purified by NH4Ac precipitation was achieved by resuspending the pellet in 75 ul of water, and adding 7.5 ul of 5M NH4Ac from Ambion plus 210 ul of 100% ethanol. The mixture was placed at -20C or -80C for at least one hour then centrifuged at 12000xg for 15min at 4C. The supernatant was removed and the pellet was washed in cold 70% ethanol. The samples were centrifuged at 12000xg for 5 min at 4C then all supernatant was removed. The pellet was air-dried and resuspended in DEPC-water to have a 1ug/ul concentration. The RNA was quantified by spectrophotometer (Nanodrop, Witec). Of this total RNA, 2.5 ug was amplified via the aRNA MessageAmp kit (Ambion, 1750) or 1 ug using aRNA MessageAmp II (Ambion, 1751) using according to manufacturer’s instructions. All reactions were performed at ? volumes which were brought up to the full requested volumes for the purifications with water. Any necessary volume reductions were achieved using a speedvac.
Amplification, Extracted product
All experiments using protocol P-MEXP-9260: (E-CAGE-189, E-CAGE-190, E-CAGE-51, E-CAGE-52, E-CAGE-53, E-CAGE-54, E-CAGE-56, E-CAGE-57, E-CAGE-58, E-CAGE-59)