|
| Status |
Public on Apr 20, 2024 |
| Title |
M3 |
| Sample type |
RNA |
| |
|
| Source name |
Total RNA from human bile exosome samples
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: bile exosome
disease: malignant biliary obstruction
patient: patient No.3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bile samples were obtained from patients undergoing preoperative biliary drainage, collected within 6 hours after emptying the drainage bag and immediately stored at -80℃ until further processing and testing. The isolation of exosomes was conducted via differential ultracentrifugation. Total RNA was extracted using an improved TRIzol-based method.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and array hybridization were performed according to the manufacturer’s protocol (Arraystar Inc.). Briefly, 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.
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| |
|
| Hybridization protocol |
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.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned using the Agilent Scanner G2505C.
|
| Data processing |
Scanned images were imported into Agilent Feature Extraction software for raw data extraction. Quantile normalization of raw data and subsequent data processing were performed using the R software limma package. 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. When comparing two groups of profile differences (such as disease versus control), the “fold change” (i.e. the ratio of the group averages) between the groups for each circRNA is computed. The statistical significance of the difference may be conveniently estimated by t-test. circRNAs having fold changes 2.0 and p-values 0.05 are selected as the significantly differentially expressed. One can filter the analysis outputs and rank the differentially expressed circRNAs according to fold change, p-value etc, using Microsoft Excel’s Data/Sort & Filter functionalities.
The significantly differentially expressed circRNAs were defined as normalized log2 ratios>1.5 and p-values <= 0.05).
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| |
|
| Submission date |
Mar 29, 2023 |
| Last update date |
Apr 20, 2024 |
| Contact name |
Ningyuan Wen |
| E-mail(s) |
wenningyuan611@hotmail.com
|
| Organization name |
West China Hospital, Sichuan University
|
| Street address |
No.37 GuoXueXiang, Wuhou district, Chengdu, China
|
| City |
Chengdu |
| ZIP/Postal code |
610041 |
| Country |
China |
| |
|
| Platform ID |
GPL21825 |
| Series (1) |
| GSE228477 |
An exosomal circRNA signature in the diagnosis and prognostification of cholangiocarcinoma: circRNA microarray analysis for 10 human bile samples |
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