GSE126399 ( miRNA )

gastric cancer ( gastric )

Ascites-derived circulating microRNAs as potential diagnostic biomarkers of gastric cancer-associated malignant ascites

Cancer : 12 / Control : 10

The differential gene expression was classified by Benjamini-Hochberg's false discovery ratio method. GeneSpring GX12 software. Gene ontology and KEGG pathway for the differential expressed genes was analyzed using GeneSpring GX 12 (Agilent Technologies, San Carlos, CA) and DAVID Bioinformatics tools (National Institute of Allergy and Infectious Diseases, NIH).

Malignant ascites is the most relevant body fluid in which to seek diagnostic biomarkers for peritoneal carcinomatosis. We aimed to identify and validate ascites-derived circulating microRNAs (miRNAs) that are differentially expressed between liver cirrhosis-associated benign ascites (LC-ascites) and gastric cancer-associated malignant ascites (GC-ascites). MiRNA expression levels were investigated in three independent cohorts. Overall, 165 ascites samples (73 LC-ascites and 92 GC-ascites) were obtained from the National Biobank of Korea. Initially, microarrays were used to screen the expression levels of 2,006 miRNAs in the discovery cohort (n = 22). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses were used to validate the expression levels of selected miRNAs in the training (n = 70) and validation (n= 73) cohorts.

All ascites samples were transported to the National Biobank of Korea within 1 hour of collection and then centrifuged at 3,134 g for 10 min to remove large cell particles and cell debris. The supernatant from each sample was aliquoted into microcentrifuge tubes and stored at -80°C prior to miRNA measurements. Exosomal RNA was extracted from the 22 ascites samples in the discovery cohort (10 liver cirrhosis [LC]-ascites and 12 gastric cancer [GC]-ascites).

n/a

Exosome from biofluid was precipitated using miRCURY Exosome Isolation Kit (Exiqon). Exosomal RNA was isolated using Trizol LS Reagent (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer’s instructions. Total RNA was quantified by absorbance at 260 nm and the integrity of small RNA was checked by Bioanalyzer2100 (Agilent, CA, USA).

total RNA

Cy3

Cyanine 3-labeled cRNA (complementary RNA) was generated from Agilent’s Low RNA Input Linear Amplification kit with 500ng total RNA. Labeled cRNA was also measured using Nanodrop spectrophotometer.

Exosome from biofluid was precipitated using miRCURY Exosome Isolation Kit (Exiqon). Exosomal RNA was isolated using Trizol LS Reagent (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer’s instructions. Total RNA was quantified by absorbance at 260 nm and the integrity of small RNA was checked by Bioanalyzer2100 (Agilent, CA, USA).

The microarray chip was scanned using Agilent’s DNA microarray scanner and the raw signal density was acquired from feature Extraction software.