Tissue RNA Purification Kit

Tissues
Tissue from animal or plant (either fresh or frozen at -70C until use) can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle. Homogenize tissue samples in 1 ml Solution RL per 50-100 mg tissue using a tissue homogenizer or rotor-stator.

Adherent Cells
Lyse cells directly in a culture dish by adding 1 ml of Solution RL to the dish and passing the cell lysate several times through a pipet tip. The amount of Solution RL required is based on the culture dish area (1 ml per 10 cm2) and not on the number of cells present.

Suspension Cells
Harvest cells and pellet cells by centrifugation. Use 1 ml of the Solution RL per 5-10 x 106 animal, plant, or yeast cells, or per 1 x 107 bacterial cells. Lyse cells by repetitive pipetting up and down. Do not wash cells before addition of Solution RL to avoid any mRNA degradation. Disruption of some yeast and bacterial cells may require a homogenizer.
Incubate at 15-30C for 5 minutes, to lyse the nucleiprotein complex completely.
Centrifuge at 12, 000 rpm for 5 min at 4C, transfer the supernatant to a new Rnase-free microcentrifuge tube. This step can eliminate protein, fat, polysaccharide, musle or plant fibre.
Add 200 ul chloroform, mix by vortexing for 15 seconds, incubate at room temperature for 3 minutes.
Centrifuge the sample at 12, 000 rpm for 10 min at 4C.
Note: After centrifugation, the mixture separates into a lower, yellow phenol-chloroform phase, an interphase, and a colorless upper aqueous phase which contains the RNA. Transfer of the colorless, upper phase containing the RNA to a new RNase-free tube.
Add an 0.5 volume of ethanol. Mix well, a visible precipitate may form after adding ethanol. Transfer the mixture to a spin column, centrifuge at 12, 000 rpm for 30 seconds at 4C, discard the flow-through.
Add 500 ul Wash Buffer RPI (check whether ethanol is added or not), Centrifuge at 12, 000 rpm for 30 seconds at 4C, discard the flow-through.
Add 500 ul Wash Buffer RW (check whether ethanol is added or not), incubate at room temperature for 1 minutes. Centrifuge at 12, 000 rpm for 30 seconds at 4C, discard the flow-through. Repeat this step again.
Centrifuge the column at 12, 000 rpm for 2 min. Air dry the column.
Place the spin column in a clean 1.5 ml microcentrifuge tube (not provided), and pipet 30-100 ul Rnase-free water directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrifuge for 2 min at 12, 000 rpm to elute. The tube contains the purified RNA. Store the RNA at -70C.