Each PDAC sample obtained from Endoscopic Ultrasound-Guided Fine-Needle Aspiration was mixed with 100 µl of Matrigel (BD Biosciences) and injected in the upper right flank in a nude mouse (Swiss Nude Mouse Crl: NU(lco)-Foxn1nu, Charles River Laboratories). Each sample derived from surgery resection was fragmented, mixed with 100 µl of Matrigel (BD Biosciences) and implanted with a trocar (10 Gauge, Innovative Research of America, Sarasota, FL) in the subcutaneous right upper flank of an anesthetized and disinfected mouse. When tumors reached 1 cm3, mice were sacrificed and tumors were removed and immediately drawn in cold guanidinium thiocyanate to perform total RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with cold guanidinium thiocyanate (4M) solution, followed by protein denaturation and RNAs extraction according to Chirgwin’s protocol . Total RNA was them purified using the RNeasy Mini kit with DNAse digestion (Qiagen, Valencia, CA) and resuspended in PCR-grade water. To ensure adequate quality, RNA was analysed with an Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA) in the Quebec Genomics Center Microarray core facility.
Label
biotin
Label protocol
Total mRNA was RNase H reduced and labelled with the SuperScript One-Cycle cDNA Kit following manufacturer's instructions (Invitrogen).
Hybridization protocol
Fifteen micrograms of fragmented cRNA were hybridized to the Affymetrix HuGene 2.0 for 16 hrs at 45°C under constant rotation. After hybridization, chips were processed using the Affymetrix GeneChip Fluidic Station 450 (protocol FS450_0007). Staining was made with streptavidin-conjugated phycoerythrin (SAPE, Molecular Probes), followed by amplification with a biotinylated anti17 streptavidin antibody (Vector Laboratories), and followed by a second round of SAPE.
Scan protocol
Chips were scanned with a GeneChip scanner 3000 G7 (Affymetrix)
