Computational protocol: Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station

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Protocol publication

[…] Amplified RNA was processed and hybridized to the Whole Human Genome (8 × 60K) Oligo Microarray (Agilent Technologies), according to manufacturer’s protocol. Slide scanning was performed using the Agilent DNA Microarray Scanner (Agilent Technologies) by DNA Chip Research Inc. (Yokohama, Japan). Expression profiles were collected in triplicate at each time point, and scanning data were normalized with Agilent’s Feature Extraction software (Agilent Technologies). Data preprocessing and analysis were performed using the GeneSpring software 11.0.1 (Agilent Technologies). Preprocessing was performed according to manufacturer’s recommendations and MicroArray Quality Control project reports []. Briefly, a decision matrix determines whether each transcript is reliably detected (i.e., present), marginally detected (i.e., marginal), or not detected (i.e., absent), and calculates the signal intensity. Normalization was performed to the 75th percentile of each array, and each gene to the median, with GeneSpring’s normalization option. Hierarchical cluster analysis was performed using principal component analysis (PCA), rank correlation of log ratios, and condition tree clustering options of GeneSpring. The probability was 0.1 and was adjusted by the false-discovery rate for correction of multiple tests. All raw fluorescence intensity data and microarray image files were deposited within the public repository for microarray-based gene expression data, the “Gene Expression Omnibus” (GEO) (http://www.ncbi.nlm.nih.gov/geo/), complying with the minimum information requirement for microarray experiments. The GEO accession number for the current experiment is GSE74708. [...] Synthesis of cDNA (1 μg) was carried out using the PrimeScript RT reagent Kit (TaKaRa Bio, Shiga, Japan) following the manufacturer’s instructions. The primers were designed using Primer Express 3.0 (Applied Biosystems, Foster, CA). Primer pair sequences are described in . The qPCR experiment was performed with SYBR Premix Ex Taq (TaKaRa Bio) using the 7500 Real-Time PCR system (Applied Biosystems). The changes in gene expression were analyzed by the relative quantitative method in a given sample relative to the control sample, and specific mRNA transcript levels were expressed as fold difference. The results were normalized to the corresponding intensity of 18S rRNA as an internal control. Each primer/cDNA set was checked in duplicate from two independent samples. […]

Pipeline specifications

Software tools GeneSpring GX, Primer Express
Databases GEO
Applications Gene expression microarray analysis, qPCR
Organisms Homo sapiens