Human lncrna array v4

Complementary DNA (cDNA) labeled with Cy3-dCTP (for primary HCC) or Cy5-dCTP (for recurrent HCC) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction using a cRNA Amplification and Labeling Kit (CapitalBio, Beijing, China) [22 (link)]. The labeled cDNA was hybridized with CapitalBio Technology Human LncRNA Array V4 containing probes inspecting about 41,000 human lncRNAs and approximately 34,000 human mRNAs. Microarray data were analyzed using the GeneSpring software V13.0 (Agilent, Santa Clara, CA, USA). Genes with an absolute fold change value ≥2 and a Benjamini-Hochberg corrected P- value ≤0.05 were treated as differentially expressed genes. Hierarchical clustering analysis was performed using Cluster 3.0 software (Stanford University, CA, USA).

CapitalBio Technology Human LncRNA Array v4 (4 × 180 K) was used in this experiment. Total RNA was extracted from placental tissues using the TRIzol Reagent (CWBio, China). RNA purity and concentration were spectrophotometrically determined from OD260/280 readings using a NanoDrop ND-1000. RNA integrity was assessed by capillary electrophoresis using the Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit (Agilent Technologies Inc., Santa Clara, CA, USA). RNA amplification, labeling, and hybridization were conducted according to the manufacturer's instructions. Briefly, double-stranded complementary DNA was transcribed from total RNA, then synthesized into complementary RNA and labeled with a fluorescent dye (Cy3-dCTP). The labeled complementary RNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies Inc.).

The global profiling of human lncRNA and mRNA expression were performed with the CapitalBio Technology Human lncRNA Array V4 (CapitalBio, Beijing, China). The acquired tiff format images were obtained using Agilent Feature Extraction (V10.7). Quantile normalization, quality control and subsequent data processing were performed using the Agilent GeneSpring software (V13.0). The differentially expressed lncRNAs and mRNAs were identified via Volcano plot filtering with the threshold set of fold change ≥2.0 and P-value < 0.05. Further hierarchical clustering analysis was performed to present lncRNA and mRNA expression patterns.