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RNA chip expression profiling analysis of HGC-27 cells after oxymatrine intervention, Agilent Human ceRNA Microarray 2019 (4*180k, Design ID: 086188) chip

ABSTRACT: In this study, we intervened HGC-27 cells with oxidized sophoridine alkaloid (OMT) at a concentration of 2.64 mg/mL for a duration of 48 hours. Subsequently, we conducted comprehensive chip expression profiling analysis on these intervened HGC-27 cells as well as normal HGC-27 cells, encompassing mRNA, lncRNA, and circRNA. We employed the Agilent Human ceRNA Microarray 2019 (4*180k, Design ID: 086188) chip for the expression profiling. To ensure analysis reliability, we quantified the total RNA of samples initially and then assessed the integrity of RNA using Agilent Bioanalyzer 2100 (Agilent Technologies). Following RNA quality inspection, we followed a standardized procedure to label the samples and hybridize them with the chips, followed by elution. The labeling process involved reverse transcription of total RNA into double-stranded cDNA, followed by synthesis of cRNA labeled with Cyanine-3-CTP (Cy3). After hybridization of labeled cRNA with the chips, we used the Agilent Scanner G2505C (Agilent Technologies) for scanning to obtain raw images. In the data processing phase, we employed the Feature Extraction software (version 10.7.1.1, Agilent Technologies) to process the raw images and extract data. Subsequently, we performed quantile normalization on the raw data to ensure data consistency. Following normalization, we filtered the data, ensuring that at least 75% of probes from each group of samples were detected, and these retained probes were used for subsequent comparison and analysis. In essence, our study culminated in a comprehensive exploration of gene expression dynamics, regulatory RNA elements, and their potential roles in the context of HGC-27 cells under the influence of OMT intervention.

ORGANISM(S): Homo sapiens

PROVIDER: GSE242236 | GEO | 2023/12/29

REPOSITORIES: GEO

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Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65°C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturer’s instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed student’s t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%.

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RNA chip expression profiling analysis of HGC-27 cells after oxymatrine intervention, Agilent Human ceRNA Microarray 2019 (4*180k, Design ID: 086188) chip

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