Luxscan 3

The mRNA gene expression profiling of SH-SY5Y cells infected with HEV71 was carried out using the 35 K Human Genome Array (Operon), which comprised ∼70 bp oligonucleotide probes for 35035 genes from the human genome Oligodatabase (human_V4.0) (CapitalBio). Firstly, SH-SY5Y cells were transiently transfected with the miR-1246 inhibitor or the negative control using the HiPerFect Transfection Reagent (QIAGEN) according to the manufacturer’s instructions. At 12 hpi, the cells were lysed with TRIzol (Invitrogen) and frozen for mRNA profiling analysis according to the manufacturer’s protocol. All data were submitted to the GEO microarray database according to LuxScan 3.0 standards (CapitalBio). All files were transformed and normalized using Loess normalization techniques. The degree of fold-change (relative fluorescence intensity) was analyzed for all of the differentially regulated genes. The significant genes list was determined for hierarchical clustering.

To identify differences in miRNA profile between the 5-8F/Si-control and the 5-8F/Si-c-Myc cell lines, the miRNA microarray analysis was performed using a miRNA microarray obtained from from Capital Bio Corporation (Beijing, China) and composed of 434 human (containing 122 predicted miRNA sequences from a published reference), 196 rat and 261 mouse miRNAs that were registered in the Sanger miRBase miRNA database (http://www.mirbase.org/; miRBase Release 8.2). The miRNA microarray used was a single-channel fluorescence chip with all oligonucleotide probes being labelled with Cy3 fluorescent dyes (green colour). The miRNAs were enriched from total RNA-extracted cells (5-8F/Si-c-Myc and 5-8F/Si-control) using a mirVana miRNA Isolation Kit (Ambion, Foster City, CA) and labelled using a mirVana Array Labelling Kit. Labelled miRNAs were then hybridized to miRNA microarrays that had 509 probes in triplicate to determine differential expression between the cell lines. This procedure was repeated twice. Fluorescence scanning was performed using a double-channel laser scanner (LuxScan 10K/A; CapitalBio). Figure signals were transformed to digital signals using image analysis software (LuxScan3.0; CapitalBio). Raw data were normalized and analyzed using the Significance Analysis of Microarrays software (SAM, version 2.1; Stanford University, CA).

The analysis of miRNA microarray data was conducted in 32 clinical samples acquired from lung adenocarcinoma patients with BM in contrast to 55 patients without BM obtained from the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing, China) between 2003 and 2008. In short, total RNA isolated from patient samples was examined with the mammalian miRNA array V2.0 (CapitalBio, Beijing, China), which identifies 1105 miRNAs in humans, mice, and rats. Separation of low-molecular-weight RNAs from total RNA was carried out by a PEG precipitation method; the low-molecular-weight RNAs were then labeled with 5-phosphate-cytidyl-uridyl-Cy3–3 and then hybridized to the mammalian miRNA array overnight at 42 °C. A LuxScan 10 K/A laser confocal scanner was used to scan the arrays, and the acquired images were evaluated using LuxScan 3.0 software (both from CapitalBio). Cluster 3.0 was used to carry out clustering analysis, and the results were viewed with TreeView software. The normalization of fluorescence signals was carried out using the median center tool for genes in Cluster 3.0, and they were evaluated using the significance analysis of microarrays (SAM), with a false discovery rate (FDR) threshold set of 0 and fold-change established at ≥2- or ≤ 0.5-fold change and p value < 0.05.

The analysis of miRNA microarray data was conducted in 32 clinical samples acquired from LAD patients with BM in contrast to 55 patients without BM. In short, total RNA isolated from patient samples was examined with the mammalian miRNA array V2.0 (CapitalBio, Beijing, China), which identified 1105 miRNAs in humans, mice, and rats. Separation of low-molecular-weight RNAs from total RNA was conducted by the polyethylene glycol precipitation method; then low-molecular-weight RNAs were labeled with 5-phosphate-cytidyl-uridyl-Cy3-3 and hybridized to the mammalian miRNA array overnight at 42 °C. The LuxScan 10 K/A laser confocal scanner was used to scan the arrays, and the acquired images were evaluated using LuxScan 3.0 software (both from CapitalBio). Cluster 3.0 was used to conduct the clustering analysis, and the results were viewed with TreeView software.

Once the on-cassette immunoassay was completed, the fluorescent signal of the detection microarray was excited by an LED and captured by the camera. Differences in the median fluorescence intensity (MFI) between the signal and the blank were determined by LuxScan 3.0 software (CapitalBio). According to the data extracted from the images, standard curves for the three biomarkers were plotted, and the concentration of each target in the clinical samples was accurately quantified with the corresponding curves.

After probe hybridization, microarrays were scanned with a confocal LuxScan^™ scanner (CapitalBio, Beijing, Chian), and the data for the obtained images were extracted with SpotData software (CapitalBio, Beijing, China). Normalization and analysis of microarray data were performed using LuxScan 3.0 software (CapitalBio, Beijing, China). Faint spots with intensities < 400 units after background subtraction in both Cy3 and Cy5 channels were removed for individual channel data extraction. A space- and intensity-dependent normalization was performed using the LOWESS program [55 (link)]. The differentially expressed genes were determined and analyzed using a t-test method and SAM software [56 (link)]. Significantly up- or down-regulated genes were filtered with fold-change values ≥ 2 or ≤ –2, respectively, in at least one time-point with q-value ≤ 0.05 in t-test. The Treeview program was used to visualize the cluster data of the differentially expressed genes [57 (link)]. The microarray dataset has been submitted to the Gene Expression Omnibus database with an accession number of GSE74060.