Human lncrna microarray v3

A pair of chemo-resistant and parental SCLC cells was used for expression profiling. RNA extraction and microarray hybridization were performed by Kangchen Company (Shanghai, China) using a human lncRNA microarray (v.3.0; Arraystar, Inc., MD, USA). The data were analyzed by means of GeneSpring 12.6 software (Agilent Technologies, Santa Clara, CA, USA) and the raw signals were log-transformed and normalized using the percentile shift normalization method, with the value set at the 75th percentile. Data processing and statistical analysis for lncRNA data were performed, and heat maps were generated.

HUVECs were exposed to 30 or 5.5 mM D-glucose for 24 h. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies, Inc.). A NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to measure the quantity and quality of the RNA. Arraystar Human lncRNA Microarray V3.0 was designed for the global profiling of human lncRNAs. Following the isolation of rRNA (using the mRNA-ONLY™ Eukaryotic mRNA Isolation kit, Epicentre), mRNA was purified from total RNA. The RNA was amplified and transcribed into fluorescent cRNA by utilizing a random priming method (Arraystar Flash RNA Labeling kit, Arraystar). Each labeled cRNA was fragmented by the addition of Blocking Agent and Fragmentation Buffer (Agilent Technologies, Inc.), and the mixture was then heated at 60°C for 30 min. The labeled cRNA was diluted by 2xGE Hybridization buffer (Agilent Technologies, Inc.). The hybridization solution was then dispensed into the gasket slide and assembled to the lncRNA expression microarray slide for 17 h at 65°C in Hybridization Oven (Agilent Technologies, Inc.). Finally, the hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (no. G2505C; Agilent Technologies, Inc.).

Arraystar human lncRNA microarray V3.0 (Array-Star, Rockville, MD, USA) contains the transcripts from authoritative public transcription database, and was designed for the global profiling of human lncRNAs and mRNAs. The microarray work was completed by KangChen Bio-tech (Shanghai, China).
QRT-PCR was performed to determine the relative expression levels of lncRNAs and genes. Primescript RT regent kit (TaKaRa, Dalian, Japan) was used for the reverse transcriptase (RT) reaction. In brief, the RT reaction was performed for 15 minutes at 37°C, followed by 5 seconds at 85°C and 1 minute at 4°C with Prime Script RT Master Mix. The qRT-PCR was performed to quantify the expression of lncRNAs using an ABI Prism 7900 Real-Time System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (Takara). The primers used in the qRT-PCR were designed by Ribo Bio-tech (Guangzhou, China) and are shown in Table 2. β-actin was used as an internal control. The data were analyzed using the 2^−ΔΔCt (∆∆Ct = [mean Ct value of lncRNA – mean Ct value of β-actin] in the AECOPD or stable COPD subjects – mean value [mean Ct value of lncRNA – mean Ct value of β-actin] in the control subjects) method and presented as relative expression level.