Agilent bioanalyzer rna 6000 nano

The extracted RNA (100 ng) was subjected to Cy3-labeled cRNA synthesis using a Low Input Quick Amp Labeling Kit (5190-2305: Agilent Technologies). The amplified RNA and dye incorporation were quantified using the Agilent Bioanalyzer RNA 6000 Nano and hybridized to a microarray chip (SurePrint G3 Rat GE Microarray 8X60K ver. 2.0, Agilent Technologies). After hybridization, the chips were washed using the Gene Expression Wash Pack. The arrays were scanned with an Agilent SureScan G4900DA, and fluorescence intensity was extracted using Feature Extraction software ver. 11.5.1.1.
Raw data of the microarray intensities were normalized using the 75th percentile shift method with GeneSpring software ver. 14.9 (Agilent Technologies) for inter-microarray variability. Differential gene expression was defined as a two-fold change relative to that of the control. We performed Gene Ontology (GO) analysis to summarize the biological functions of the selected genes. The data were then processed using Fisher’s exact test and multiple test correction to identify significant over-representation of GO annotations belonging to the selected genes.

The total RNA was extracted from the cervical lymph nodes of the four groups using ISOGEN reagent (Nippon Gene Co. Ltd., Toyama, Japan), according to the manufacturer’s instructions. The total RNA concentration and purity were measured spectrophotometrically using a NanoDrop1000 (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was monitored by electrophoresis on an Agilent Bioanalyzer RNA 6000 Nano (Agilent Technologies, Santa Clara, CA, USA).

iPSC-CMs were cultured on GelMA substrates for 7 days. The purified iPSC-CMs were lysed with 700 μl QIAZOL (QIAGEN), following the protocol of the miRNeasy Micro Kit with RNase-Free DNase Set (QIAGEN) for RNA digestion. The concentration of eluted RNA is measured with Qubit™ RNA HS Assay Kit, and the RNA integrity number (RIN) is assessed using the Agilent Bioanalyzer RNA6000 Nano. RNA sequencing was conducted by Guangzhou Gene Denovo Biotechnology Co., Ltd. (Guangzhou), and bioinformatic analysis was performed using Omicsmart, a dynamic and interactive online platform for data analysis (https://www.omicsmart.com).