Human microrna microarray

Microarray Expression data are Agilent Human MicroRNA microarray and come from a study by Martin-Pérez et al. (26 (link)) and data for lymph nodes (LN) (GSE23026) from the same research team. Data were processed and analyzed in R environment (24 (link)), using package AgimicroRNA (27 ) for processing microarrays and limma (28 ) for differential analysis. Thus, the workflow would be: (i) Preprocessing microarray using background method ‘half’, which establishes a value for negative data. (ii) Quantile normalization (iii) and default filtering method removing control and non-quality probes. (iv) An experimental Bayes moderated t-statistics test was carried out from limma package in order to observe differential expression in such data.

The serum RNA samples from the 52 ESCC patients and 52 healthy controls were assayed for miRNA expression profiles by using the Agilent Human microRNA microarray, which contains the probes for 2006 human miRNAs. Briefly, 100 ng total RNA was labeled with Cy3 by using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Each slide was hybridized with 100 ng Cy3-labeled RNA in a hybridization oven (Agilent Technologies, Santa Clara, CA, USA) at 55°C, 20 rpm for 20 h according to the manufacturer's instructions. After hybridization, slides were washed with the Gene Expression Wash Buffer Kit (Agilent Technologies, Santa Clara, CA, USA) in staining dishes (Thermo Shandon, Waltham, MA, USA). The washed slides were scanned and the microarray images were then converted into spot intensity values by the Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA). The signals undergone background subtraction were exported directly into the Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, USA) with default settings. Raw data were normalized by the quartile algorithm with the Gene Spring Software 12.6 (Agilent Technologies, Santa Clara, CA, USA).

Human microRNA Microarrays (Release 21.0; Agilent Technologies Ltd., Santa Clara, CA, USA) containing 2,568 mature microRNA sequences were performed for preliminary screening of dysregulated circulating plasma microRNAs in NSCLC patients with different EGFR mutation genotypes. Raw data were extracted using Agilent's Feature Extraction software (version 10.7; Agilent Technologies Ltd., Santa Clara, CA, USA) and normalized using GeneSpring software (Agilent Technologies Ltd., Santa Clara, USA) with the normalization of labeling spike-in RNA and hybridization spike-in RNA according to the manufacturer's protocol.

Human microRNA microarrays from Agilent Technologies (8*60 K) containing probes for 2549 human microRNAs from the miRBase V21.0 database were used. The microarray image information was converted into spot intensity values using Scanner Control Software Rev. 7.0 (Agilent Technologies, Santa Clara, CA). Raw data were normalized by Quantile algorithm, included in the R package AgiMicroRna [27 (link)]. The microarray experiments were performed at Shanghai Biotechnology Corporation and microRNAs with altered expression levels were screened in each experimental group according to the manufacturer’s protocol. The screening cohort included 5 type 2 DM patients, 6 DN patients, and 9 NDRD (4 IgAN and 5 MN) patients.