strain background: C57Bl6 cell line origin: B16 cell type: highly metastatic melanoma cell line B16 variants
Treatment protocol
B16F1 and B16-variants were cultured in DMEM (Gibco BRL, Grand Island, NY) supplemented with 20% fetal bovine serum. B16F1 cells (1x106) were injected intradermally in the backskin of C57BL6 mice and the tumor was removed 14-17 days after implantation. Metastatic melanoma cells were isolated from lymph nodes (LN) and cultured in vitro (B16-RI). Then, B16-RI was again intradermally injected into mice, and primary tumors, LN metastases (B16-RII) and lung metastases (B16-RIIL) were isolated and re-cultured. Funcitonal assays were performed on B16 variants in vitro.
Growth protocol
B16variants were seeded onto culture dishes (10 µg/ml; BD Biosciences, Bedford, MA) and were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY), 2 mM L glutamine, antibiotic-antimycotic solution for up to 10 passages.
Extracted molecule
total RNA
Extraction protocol
B16 and B16 variants were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) supplemented with antibiotic-antimycotic solution. Every B16 variant was cultured in duplicates. Total cellular RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) extracted with chloroform, precipitated with isopropanol, washed with 70% ethanol, and dissolved in DNase-free/RNase-free distilled water. The concentration of RNA was measured using NanoDrop ND-1000 spectrophotometer (Witec AG, Littau, Switzerland) and RNA quality was assessed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Chemiluminescent Digoxigenin-UTP labeled cRNA
Label protocol
Digoxigenin-UTP labeled cRNA was generated from 0.5 µg of total RNA for each sample using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit (P/N 4363105) according to the manufacturer’s protocol.
Hybridization protocol
Hybridization protocol Array hybridization, array processing, chemiluminescence detection, image acquisition, and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following manufacturer’s protocol.
Scan protocol
Scan protocol Chemiluminescent Detection Kit (P/N 4339627) and Chemiluminescent Microarray Analyzer User Guide (P/N 4338852B).
Description
R2 A
Data processing
Data processing The Expression Array System Software suite performs the auto-gridding, feature extraction, fluorescence normalization, and signal data generation. The quantification data contain many distinct measurements for each probe, including the three basic measurements: Signal, S/N, and Flag values. The Signal value is the fully corrected, background subtracted measurement of chemiluminescent signal for gene expression values. The S/N value represents the ratio of signal above noise, or the measurement uncertainty of the probe signal, and can be used as a confidence level for the probe measurement. An S/N of 3 represents approximately a 99.95% confidence that the probe is detected above the background noise. In situations where the probe showed S/N < 1, the signal measurement is replaced with a 1 SDEV upper limit based on its probe signal SDEV.