UM_TotalRNA_NucleoZOL(15)

user's manuals about RNA or or

5.3 Isolation of total RNA in combination with NucleoSpin®

RNA Set for NucleoZOL

1

Homogenization

The homogenization is similar to the standard protocols. For detailed information refer to section 5.1 or e up to 50 mg tissue with 500 μL NucleoZOL. Larger samples may exceed the RNA binding capacity of the NucleoSpin® RNA Binding Column. Use up to 5 x 106 cells with 500 μL NucleoZOL per preparation. Use up to 200 μL liquid sample with 500 μL NucleoZOL.2

Precipitate contaminants

Add 200 μL RNase-free water/500 μL NucleoZOL to the lysate.

Vortex the sample vigorously for 15 s and incubate at room temperature for 5–15 min.

For samples containing 50 mg tissue/500 μL 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 500–600 μL supernatant into a fresh tube (not provided). Do not disturb the DNA/protein pellet.

The pellet containing DNA, protein, and polysaccharides comprises approximately 10 % in volume of the total homogenate-water mix.3

Adjust RNA binding conditions

Add 500 μL Buffer MX to the sample homogenate and mix by vortexing.Add 1 vol Buffer MX per 1 mL NucleoZOL used for homogenization.