Cachectic transgenic mice were defined as Pdx1-cre;LSL-KrasG12D;INK4a/arffl/fl mice, developing systematically spontaneous pancreatic adenocarcinoma and died between 8 and 12 weeks-old. Animals were obtained by crossbreeding the following strains: Pdx1-Cre (Gu et al, 2002) (kindly given by D. Melton), LSL-KrasG12D (Aguirre et al, 2003) and INK4a/arffl/fl (Aguirre et al, 2003; Bardeesy et al, 2006) (kindly given by R. Depinho, Dana-Faber Cancer Institute, MA). Mice were kept within the Experimental Animal House of the Centre de Cancérologie de Marseille (CRCM) pole Luminy, following institutional guidelines.
Extracted molecule
total RNA
Extraction protocol
Liver, intra-abdominal adipose and biceps femoris muscle tissues were collected from cachectic Pdx1-cre;LSL-KrasG12D;INK4a/arffl/fl or LSL-KrasG12D;INK4a/arffl/fl control mice (10 weeks-old) and immediately drawn in cold guanidinium thiocyanate (4 M) solution, followed by protein denaturation and RNAs extraction according to Chirgwin’s protocol (Chirgwin et al, 1979). Total RNA was purified on RNeasy mini kit column (Qiagen, Mississauga, ON, Canada) before labeling protocol.
Label
Biotin
Label protocol
Chips were processed with total RNA (200 ng per sample) according to the WT Expression kit for Affymetrix Whole Transcrit Expression Arrays protocol (Ambion, Austin, TX ), labeled using the WT Terminal Labeling and hybridized to the arrays as described by the manufacturer (Affymetrix, Santa Clara, CA).
Hybridization protocol
The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After 17±1h of hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol (http://media.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf).
Scan protocol
The arrays were scanned using the Affymetrix GCS 3000 7G to produce the intensity files and the image data were analyzed by using the Expression Console Software for quality control (www.affymetrix.com).
Data processing
Background subtraction and normalization of probe set intensities were performed using the method of Robust Multiarray Analysis (RMA) described by Irizarry et al. (Irizarry et al, 2003). Microarray analyses were performed by the CHU de Québec Research Center (CHUL) Gene Expression Platform, Quebec, Canada.
