GSE112496 ( circRNA )

Esophageal Squamous Cell Carcinoma ( esophageal )

Microarray expression profile and functional analysis of circular RNAs in plasma for Esophageal Squamous Cell Carcinoma

Cancer : 5 / Control : 5

Scanned images were imported into Agilent Feature Extraction software for raw data extraction. After quantile normalization of the raw data, low intensity filtering was performed, and the circRNAs that at least 5 out of 10 samples have flags in “P” or “M” (“All Targets Value”) were retained for further analyses.

The purpose of this study is to explore the circRNAs expression profiles in the plasma from esophageal squamous cell carcinoma (ESCC) patients.The purpose of this study is to explore the circRNAs expression profiles in the plasma from esophageal squamous cell carcinoma (ESCC) patients.

We have completed the Arraystar Human circRNA Array analysis of the 10 plasma samples from preoperative ESCC patients (n=5) and healthy controls (n=5).

Shen Y, Shao Y, Niu C, Ruan X et al. Systematic Identification of circRNA-miRNA-mRNA Regulatory Network in Esophageal Squamous Cell Carcinoma. Front Genet 2021;12:580390. PMID: 33747034

Total RNA was extracted from 1mL plasma using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacture’s protocol. The integrity of RNA was evaluated by 1% denaturing agarose gel electrophoresis. The concentration and purity of total RNA were then measured using the NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA).

total RNA

Cy3

Total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.

1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven

The hybridized arrays were washed, fixed and scanned using the Agilent Scanner G2505C.