UM_TotalRNA_NucleoZOL(13)

user's manuals about RNA or or

2

Precipitate contaminants

Add 400 μL RNase-free water per 1 mL NucleoZOL to the lysate. Shake the sample vigorously for 15 s. Incubate at room temperature for 5–15 min. For samples containing 100 mg tissue/mL NucleoZOL, 15 min incubation at room temperature is recommended.

Centrifuge samples for 15 min at 12,000 x g. Centrifugation can be performed at 4–28 °C.

A semi-solid pellet containing DNA, proteins and polysaccharides forms at the bottom of the tube. The RNA is still solubilized in the supernatant.

Transfer 1 mL supernatant (75 % of total supernatant volume) to a fresh tube. Leave a layer of the supernatant above the DNA/protein pellet.

The pellet containing DNA, protein, and polysaccharides comprises approximately 10 % in volume of the total homogenate-water mix (e.g., about 8 % pellet for 80 mg tissue lysed in 1 mL reagent).3

Phase separation (optional)

The basic protocol for total RNA isolation can be complemented by an optional phase separation. This is useful for samples with high DNA content and/or extracellular material.

Add 5 μL (0.5 % of supernatant volume) 4-bromoanisole to 1 mL transferred supernatant. Mix well for 15 s and incubate at room temperature for 3–5 min. Do not substitute 4-bromoanisole with bromchloropropane or chloroform!Centrifuge for 10 min at 12,000 x g (4–25 °C).

Residual DNA, proteins, and polysaccharides accumulate in the organic phase at the bottom of the tube. RNA is still solubilized in the supernatant. 4

Precipitate total RNA

Pipette RNA containing supernatant from step 2 or 3 into a fresh tube.

Add 1 mL of isopropanol per 1 mL supernatant in order to precipitate RNA. Incubate samples at room temperature for 10 min.Centrifuge samples for 10 min at 12,000 x g.

Typically, RNA is obtained as a white pellet at the bottom of the tube. For spleen samples, RNA forms a gel-like membrane on the bottom of the tube. Upon washing with ethanol, the membrane becomes more visible.