P-MEXP-2387 - P-MEXP-2387

Plant tissue was ground to fine powder using liquid nitrogen in a 2mL eppendorf tube. The samples were homogenized in TRIzol Reagent (Invitrogen) : 1 mL for 50-100 mg of plant tissue, by vortexing and incubated at room temperature for 5 min. Chloroform (200µl/1mL TRIzol) was added and the capped tubes were hand-shaken for 15 sec. They were incubated for 2-3 min, then centrifuged for 15 min at 11000 rpm at 4°C. The aqueous phase was transferred to a new 1.5 mL tube and 500ul of isopropanol was added. The samples were incubated for 10 min at room temperature then centrifuged for 10 min at 11000 rpm at 4°C. The supernatant was removed and the pellet was washed with 1 mL of 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 min at 11000 rpm at 4°C. The pellet was air-dried for 10 min at room-temperature, then resuspended in 500 µl DEPC-treated water, and precipated with 2 volumes of 100 % ethanol and 1/10 V of Sodium acetate 3M, pH5,2. The samples were precipitated 30 min at –20 °C and centrifuged for 15 min at 10000 rpm at 4°C. The supernatant was removed, the pellet was air-dried and resuspended in DEPC-treated water. Quality assessment was performed with an Agilent Technologies Bioanalyser 2100 and the Ribogreen method. 2-5 µg of total RNA was amplified to aRNA using the Ambion MessageAmp Kit (Ambion, #1750).
Amplification, Extracted product
All experiments using protocol P-MEXP-2387: (E-CAGE-11, E-CAGE-12, E-CAGE-13)