tissue: lung adenocarcinoma genotype/variation: cRaf overexpressing gender: male
Treatment protocol
SP-C/c-raf transgenic mice were obtained from the laboratory of Prof. Ulf Rapp (University of Würzburg, Germany). Lung tissue from n=6 male and n=6 females SP-C/c-Raf transgenic mice (aged 4–6 months) and n= 6 male and n= 6 female normal lung tissue from type mice (aged 4-6 months) were sacrificed and the lung tissues were immediately frozen on dry ice and stored at - 80° until further analysis.From each frozen lung tissue 10-mm thick sections were prepared and transferred on polyethylene napthalate foil-covered slides (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany). The sections were fixed in methanol/acetic acid and stained in hematoxylin. The desired cells were microdissected using the PALM MicroLaser systems (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany) and collected in an adhesive cap (Zeiss, P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany). Microdissected cells were resuspended in a guanidine isothiocyanate containing buffer (RLT buffer from RNeasy MikroKit, Qiagen, Santa Clarita, CA, USA) with 10 ml/ml ß-mercaptoethanol to ensure isolation of intact RNA.
Extracted molecule
total RNA
Extraction protocol
Total RNA-extraction was performed with the RNeasy Micro Kit (RNeasy MicroKit Qiagen, Santa Clarita, CA, USA) according to the manufacturer’s instruction.
Label
Cy3
Label protocol
One hundred nanograms of total RNA samples was dephosphorylated, 3′ end-labeled with Cy3-pCp, purified on Micro Bio-Spin columns, dried, and hybridized onto arrays using the miRNA Microarray System labeling kit V2 according to the manufactures instructions (5190-0456)
Hybridization protocol
Purified Cy3 labeled RNA was hybridized accordingly to the manufacturer protocol. 20 Rotation x min 16h 60C
Scan protocol
Hybridized microarray slides were scanned with an Agilent DNA Microarray Scanner G2505C with default protocol.
Data processing
The scanned images were analyzed with Feature Extraction Software 8.1.3 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. In order to normalize data was used Quantile normalization funcion.
