Low input quick amp wt labeling kit

The microarray experiments were performed by Shanghai Biotechnology Corporation (SBC) following the protocol of Agilent Technologies Inc. (CA, USA). Briefly, total RNA was isolated and purified using a mirVana™ miRNA Isolation Kit (Ambion, TX, USA), according to the manufacturer’s instructions. RNA samples from each group were then used to generate labeled complementary RNA (cRNA) targets using the Low Input Quick Amp WT Labeling Kit (Agilent Technologies, CA, USA) for the SBC human ceRNA microarray (4 × 180 K). The labeled cRNA targets were then hybridized with the slides and the slides were scanned on the Agilent Microarray Scanner (Agilent Technologies, CA, USA). Data were extracted with Feature Extraction software 10.7 (Agilent Technologies, CA, USA). CircRNAs with high expression abundances, expression levels with more than 2-fold alteration, and a P < 0.01 were selected for further analysis.

The microarray work was performed by Shanghai Biotechnology Corporation, Shanghai, China. In brief, total RNA from each sample was amplified and labelled by Low Input Quick Amp WT Labeling Kit (Agilent Technologies), following the manufacturer's instructions. The labelled cRNA was purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labelled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop 2000. Each microarray slide was hybridized with 1.65 μg Cy3‐labelled cRNA using Gene Expression Hybridization Kit in Hybridization Oven, according to the manufacturer's instructions. After 17‐hour hybridization, slides were washed in staining dishes using the Gene Expression Wash Buffer Kit following the manufacturer's instructions. Next, the slides were scanned by Agilent Microarray Scanner G2565C (Agilent Technologies) with default settings, Dye channel: Green, Scan resolution = 3 μm, PMT 100%, 20 bit. Agilent Feature Extraction software (version 10.7) was used to analyse the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring Software 11.0 (Agilent Technologies). Differentially expressed genes (DEGs) were identified through fold change (greater than 2‐fold) filtering and with FDR adjusted P values < .05.

Total RNA was isolated using TRIzol reagent (Invitrogen). Qualified total RNA samples were amplified by cDNA according to the Low Input Quick Amp WT Labeling Kit (Agilent) and standard operating procedures (SOPs). The expression profile of lncRNA in five malignant glioma and five NBT were screened by using lncRNA microarray (Shanghai Biotechnology Corporation). Tissue sample preparation and microarray hybridization were separately performed according to manufacturer's recommendations. qRT‐PCR was used to further determine the nuclear/ cytoplasmic level of lncRNA HOXA‐AS3 in glioma cells. GAPDH and U6 were used as a nuclear and cytoplasmic control. The primer sequences for the amplification of the lncRNA HOXA‐AS3 and GAPDH used were listed in Table S1

For microarray analysis 0.2 µg of RNA were transformed to cRNA using Low Input Quick Amp WT Labeling Kit from Agilent. cRNA was labeled with Cy3 and labeled cRNA was applied to 8×15K arrays Agilent arrays. Signal intensities for probes were obtained from the scanned microarray image using Agilent Technologies' Feature Extraction software and quantile normalized. Differentially expressed genes were selected using Limma [91] (link) implemented in One Channel GUI with a p<0.01 and at least 2.5 fold change. Gene groups differentially expressed in different genotypes were identified using GSEA tool [32] (link) using hand-compiled gene lists (Table S8) that include functional categories from cyanobase, GO annotation and literature curated gene list (see supplementary material). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE51671. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ebcpyuqknbytvul&acc=GSE51671).