tissue: Pleura/pleural effusion, lymphocyte, myeloid, monocyte altered gravity condition: 1g in flight control
Treatment protocol
Human U937 cells were exposed to altered gravity conditions during a parabolic flight.
Growth protocol
U937 cells were cultured in RPMI 1640 medium (Biochrom/MerckMillipore, Germany), supplemented with 10% fetal bovine serum (FBS Superior; Biochrom/Merck Millipore, Germany), 2mM glutamine (Gibco/Life Technologies, Germany), and 100U/mL penicillin as well as 100 ug/mL streptomycin (Gibco/Life Technologies, Germany). Cells were seeded with a density of 0.2 × 10e6 cells/mL and the medium was exchanged every 48 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Midi and Maxi kit (Qiagen, Germany) The RNA quality and concentration integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
Label
Cy3
Label protocol
Cy3-labeling with the “Low RNA Input Linear Amplification Kit, PLUS, One-Color” (Agilent Technologies)
Hybridization protocol
Hybridization for 17.5 h to a NimbleGen expression microarray (12 × 135,000 features) employing the “Gene Expression Hybridization Kit” (Agilent Technologies)
Scan protocol
Scanning was performed using the Microarray Scanner G2505B (Agilent Technologies)
Description
1g IF
Data processing
The image files of the scanner were analyzed with the NimbleScan Software 2.6 using the robustmultiarray analysis (RMA) with the default parameters. RMA, a probe-level summarization method, identifies probes that are outliers in the overall behavior of the expression measured for a given gene. The contribution of outlier probes is reduced in the reported gene expression level, which has been demonstrated to improve the sensitivity and reproducibility of microarray results. In addition to screening outlier probes, NimbleScan software’s implementation of RMA [Irizarry et al., 2003] used quantile normalization and background correction.( R. A. Irizarry, B.Hobbs, F.Collin et al., “Exploration, normalization, and summaries of high density oligonucleotide array probe level data,” Biostatistics, vol. 4, no. 2, pp. 249–264, 2003)
