Agilent one color microarray based gene expression analysis protocol

Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs were measured by NanoDrop ND-1000 spectrophotometry (Thermo Scientific). 1μg of each labeled cRNA was fragmented by adding 5μl 10× blocking agent and 1μl of 25× fragmentation buffer, then heated the mixture at 60°C for 30 min, finally 25μl 2× GE Hybridization buffer was added to dilute the labeled cRNA. 50μl of hybridization solution was dispensed into the gasket slide and assembled to the lncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent Microarray Scanner.

The mRNA expression profiles of knee OA cartilage tissue were obtained from a previous study [16 (link)]. The knee cartilage specimens were collected from 6 knee OA patients (1 male/5 females) and 5 normal control (3 males/2 females). Each cartilage specimen was disserted into two groups, one for microarray experiment and the other for quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation. The extracted mRNA was purified, amplified, and transcribed into fluorescent cRNA after removing rRNA from the total RNA. The quality of cRNAs was measured using the Nanodrop ND-1000. Then, cRNA was hybridized to the Human lncRNA Array following the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Significant regulated mRNAs were defined as fold change (FC) ≥ 2.0 and P value < 0.05. Detailed information of study samples, study design, and statistical analysis could be found in Additional file 1: Table S1 and the published study [16 (link)].

Gene chip expression analysis was performed by Kangcheng Biotechnology Co., Ltd. using the Agilent array platform. First, the RNA quantity and quality were assessed using a NanoDrop ND‐1000 UV Spectrophotometer (Implen). RNA integrity of each sample was identified by standard denaturing agarose gel electrophoresis. Second, sample labeling and chip hybridization were performed according to the Agilent One‐Color Microarray‐Based Gene Expression Analysis protocol (Agilent). The samples were labeled by using the Agilent Quick Amp Labeling kit and hybridization experiments were performed by using the Agilent SureHyb. Specifically, the total RNA of each sample was linearly amplified and labeled with Cy3‐UTP; the labeled complementary RNAs (cRNAs) were purified by using the RNeasy Mini Kit (Qiagen), and the concentration and activity were detected with a NanoDrop ND‐1000 UV spectrophotometer. The purified Cy3‐UTP‐labeled cRNAs were hybridized with Agilent mouse 4 × 44 K gene expression profiling chip v2 (Agilent). The hybridization chip was washed, fixed, and scanned by using Agilent DNA Microarray Scanner (part number G2505C); Chip probe signal values were acquired by using Agilent Feature Extraction software (v11.0.1.1) to obtain raw data. Finally, the quantile normalization of raw data and subsequent data processing were performed by using GeneSpring GX v12.1 software (Agilent).