sample type: Cell line derived from pancreatic ductal adenocarcinoma cell line: Capan-1 treatment: control
Treatment protocol
Transfections were performed following the manufacturer’s protocols of the TriFECTa Dicer- Substrate RNAi kit (IDT, Coralville, IA, USA) and Lipofectamine RNAi Max Reagent (Invitrogen Corporation, Carlsbad, CA, USA). Cells were transfected with a HOXB7-specific siRNA at a final concentration of 10 nM. The mRNA content was measured 48 hours after transfection by RT-qPCR. Each experiment was repeated at least twice.
Growth protocol
Human pancreatic cancer cell line MIA PaCa-2 was obtained from American Type Culture Collection (ATCC® Number: CRL-1420™, Manassas, VA, USA). The cells were maintained routinely in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen Corporation), 100 U/mL penicillin G (Invitrogen Corporation), and 0.1 mg/mL streptomycin sulfate (Invitrogen Corporation) at 37°C in a humidified, 5% CO2, 95% air atmosphere. Capan-1 cell line established from a hepatic metastasis of a PDAC was also obtained from ATCC (Number: HTB-79™). The cells were grown in IMDM medium (Invitrogen Corporation) supplemented with 20% FBS (Invitrogen Corporation).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Duesseldorf, North Rhine-Westphalia, Germany) according to manufacturer's guidelines.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions, followed by RNAspin column purification (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Dye incorporation and cRNA yield were checked with the Nanovue Spectrophotometer (GE Healthcare).
Hybridization protocol
1.65 µg of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µL containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 55 µL of 2x GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Human GE 4x44K v2 Microarray (G2519F) for 17 hours at 65 ° C rotating at 10 rpm. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute at 37°C with GE Wash buffer 2 (Agilent), 10 seconds with acetonitrile, and 30 seconds with drying solution.
Scan protocol
Slides were scanned immediately after washing on the High-Resolution Microarray Scanner (Agilent, Santa Clara, CA, USA) using one color scan setting for 1x44k array slides.
Description
Gene expression (parental line)
Data processing
The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted by Agilent Feature Extraction software version 9.5.3. Among the spots present in each array only those with none flag (i.e. low intensity, saturation, controls, etc.) were selected for analysis.