Microarray expression profiling in the denervated hippocampus identifies long noncoding RNAs functionally involved in neurogenesis

RNA samples were extracted from the paired rat untreated hippocampus 7 days after the FF transection. The general RNA quality was evaluated by RIN. RNA quantity and purity were assessed using K5500 micro-spectrophotometer. A260/A280 ≥1.5 and A260/A230 ≥1 indicated acceptable RNA purity, and RIN value ≥7 using the Agilent 2200 RNA assay indicated acceptable RNA integrity (Table. S1). Genomic DNA contamination was evaluated by gel electrophoresis .Three independent samples were assayed to evaluate the reproducibility of the experimental procedure. Using the microarray expression profiles, 103 (74 activated and 29 repressed) lncRNAs differentially expressed in the hippocampus after the FF transection were identified. The hippocampal tissue (denervated and untreated) from three paired Sprague–Dawley rats was quickly harvested on ice. For distant shipping, all the tissues were immediately frozen in liquid nitrogen. Total RNA was then extracted with TRIzol reagent following the manufacturer’s instruction. The RNA integrity number (RIN) was evaluated to judge the integrity of RNA samples using Agilent 2200 Bioanalyzer (Agilent Technologies, USA) following the manufacturer’s protocol. The purity of RNA samples was evaluated by agarose gel electrophoresis and ultraviolet spectrophotometer K5500 (Beijing Kaiao Technology Development Co., Ltd, China). Fluorescent complementary DNA (cDNA) was synthesized from about 2 µg of total RNA using an Amino Allyl MessageAmp II Kit (Life Technologies, USA). Hybridization with RiboArrayTM lncDETECTTM RAT Array 1*12K was performed following the manufacturer’s instruction (Ribo, China). After washing, the slides were scanned and analyzed with GenePix 4000B Microarray Scanner (Molecular Devices, USA). The microarray data was corrected by 50% scaling method to eliminate the system error of the experiment. Fold change was calculated by 2-Fold change=log2 (normalized intensity of treat/normalized intensity of control).