MagAttract Direct mRNA M48 Kit (192)

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Publication protocol

Isolation of total RNA and poly(A)+ RNA: Total RNA was isolated from the tissue using the RNeasy Midi kit (Qiagen, Hilden, Germany). A cube of approximately 4 mm square was cut out from the tissue sample and placed in 1.5 ml RTL buffer, containing β-Mercaptoethanol. Three 3.2 mm Chrome-Steel beads (BioSpec Products, Inc. Bartlesville OK, USA), prewashed in RTL buffer, were added to each tube. The tissue samples were homogenized using the Mixer Mill 300 (Qiagen, Hilden, Germany) for 5 min at 30 Hz, the tube racks were rotated 180° and then homogenized for additional 5 min at 30 Hz. Normally, this was sufficient for complete homogenisation of the tissue, but additional shaking was needed for some samples. After the homogenisation, total RNA was isolated according to the manufacturer's instructions and eluted in two times 150 μl RNase-free water. Total RNA samples were concentrated using the RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany) and eluted in 20 μl RNase-free water. Subsequently, total RNA was treated with a TURBO DNase kit following the protocol (Ambion, Austin TX, US). Poly(A)+ RNA (mRNA) was isolated from total RNA using an automated BioRobot M48 workstation and the MagAttract Direct mRNA M48 kit (Qiagen, Hilden, Germany) and concentrated as described above. mRNA concentrations were measured using a BioPhotometer v. 1.32 (Eppendorf, Hamburg, Germany).

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