P-EMBL-82 - P-EMBL-82
About 13000 cells from each fraction were lysed and total RNA isolated using the Arcturus PicoPure RNA-Isolation Kit (Arcturus, Mountain View, USA). DNase treatment was performed (Quiagen, Hilden, Germany). RNA quality was controlled with the RNA 6000 Pico LabChip kit (Agilent, Waldbronn, Germany). Linear amplification of mRNA was performed by in vitro transcription over two rounds using the Arcturus RiboAmp Kit (Arcturus, Mountain View, USA). The amplified RNA was analyzed by the RNA nano Lab Chip kit (Agilent) and by the SpectraMAX plus photometer (Molecular Devices, Sunnyvale, USA) at 260 nm. We obtained about 80 µg aRNA with a continuous spectrum of RNA length of 200 to 700 base pairs (results not shown).
About 6 µg of aRNA were incubated with 3 µg Random Primer (Invitrogen, Karlsruhe, Germany) and labeled by amino-allyl-coupling using the Atlas Glass Fluorescent Labelling Kit (Clontech, Palo Alto, USA) and Cy3-monofunctional reactive dye (Amersham Biosciences, Little Chalfont, England).
Amount of nucleic acid labeled, Amplification, Label used