cell line: A549 cell line derived from human alveolar type 2 primary lung adenocarcinoma cells treatment: IR time: 24 hr
Treatment protocol
Cells were seeded on Petri dishes (35x15 or 60x15 mm), with or without glass coverslips, kept on ice before and after irradiation, and cultured at 37° C in fresh medium thereafter. Irradiation with gamma-rays was performed at the Department of Oncological and Surgical Sciences of Padova University with a 137Cs source (dose rate of 2.8 Gy/min) within the dose range 0-6 Gy. Except for irradiation, the control cells were subjected to the same experimental conditions.
Growth protocol
All culture medium were from Life Technologies (Carlsbad, CA, USA) and were supplemented with heat inactivated fetal bovine serum (FBS, BIOCHROM, Berlin, Germany), 38 units/mL streptomycin, and 100 units/mL penicillin G. All cells were grown at 37◦C in a humidified atmosphere with 5% CO2. Non-small cell carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). A549 cells derived from human alveolar type 2 primary lung adenocarcinoma cells (CCL-185™) and were cultured in Ham’s F12-K Nutrient Mixture. A549 cells were grown in a 1:1 mixture of DMEM and Ham’s F-12 medium (DMEM/F-12, Gibco, Life Technologies), HEPES 20 mM, 1% of MEM non-essential amino acids and 10% of heat-inactivated FCS. Cells were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO2, and maintained in exponential and asynchronous phase of growth by repeated trypsinization and reseeding prior to reaching subconfluency.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from irradiated and non-irradiated cells by using Trizol® Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. Total RNA quantification was performed using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA); RNA integrity and the content of miRNAs were assessed by capillary electrophoresis using the Agilent Bioanalyzer 2100, with the RNA 6000 Nano and the small RNA Nano chips, respectively (Agilent Technologies, Palo Alto, CA, USA). Only total RNA samples with RNA Integrity Number (RIN) values ≥ 6 were used for microarray analysis.
Label
Cy3
Label protocol
total RNA (200 ng) was labeled with pCp Cy3, according to the Agilent Technologies protocol ( the miRNA Microarray System protocol v. 1.5) and unincorporated dyes were removed with MicroBioSpin6 columns (BioRad).
Hybridization protocol
Probes were hybridized with labelled RNA at 55°C for 22 hours using the Agilent’s Hybridization Oven that is suited for bubble-mixing and microarray hybridization processes.
Scan protocol
Slides were washed by Agilent Gene expression wash buffer 1 and 2 and scanned using an Agilent microarray scanner (model G2565CA) at 100% and 5% sensitivity settings. Agilent Feature Extraction software version 10.5.1.1 was used for image analysis.
Description
miRNA expression profiling of A549 irradiated cells (2Gy-24h-3)
Data processing
Inter-array normalization of expression levels was performed with cyclic Lowess for miRNA experiments and with quantile for gene expression profiling [Bolstad BM et al., Bioinformatics (2003), 19(2):185-93] to correct possible experimental distortions. Normalization function was applied to expression data of all experiments and then values of spot replicates within arrays were averaged. Furthermore, Feature Extraction Software provides spot quality measures in order to evaluate the goodness and the reliability of hybridization. In particular flag ‘‘glsFound’’ (set to 1 if the spot has an intensity value significantly different from the local background, 0 otherwise) was used to filter out unreliable probes: flag equal to 0 will be noted as ‘‘null’’. So, in order to make more robust and unbiased statistical analysis, probes with a high proportion of ‘‘null’’ values were removed from the dataset. We decided to use about 40% of null as threshold in the filtering process obtaining a total of 333 available human miRNAs.
