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| Status |
Public on Mar 01, 2018 |
| Title |
Patient 3 3T_3N |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
adjacent normal
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| Organism |
Homo sapiens |
| Characteristics |
tissue: adjacent normal
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| Extracted molecule |
total RNA |
| Extraction protocol |
UHRR was bought from Agilent,HBRR was bought from Ambion.tumor samples and normal tissues were collected from the Oncology Department of the Second Affiliated Hospital of Zhen Zhou University by related surgeons. Plasma samples were isolated by EDTA and centrifuged at 820 ×g for 10 min. The postoperative samples were collected 2 to 34 days after surgery.
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| Label |
Cy5
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| Label protocol |
5ug isolated total RNA was treated with Rnase R (Epicentre, Madison, WI) to remove linear RNA. cDNA labeled with a fluorescent dye (Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction41, and the procedure has been improved by using CapitalBio circRNA Amplification and Labeling Kit (CapitalBio, Beijing) for producing higher yields of labeled cDNA. In detail, double-stranded cDNAs (containing the T7 RNA polymerase promoter sequence) were synthesized from 0.2 μg total RNA using the CbcScript reverse transcriptase with cDNA synthesis system according to the manufacturer’s protocol (Capitalbio, Beijing) with the T7 Oligo (dN). After completion of double-stranded cDNA (dsDNA) synthesis using DNA polymerase and RNase H, the dsDNA products about 12.5μL were subjected to 40μL in vitro transcription reactions at 37℃ for 14 h using a T7 Enzyme Mix. The amplified cRNA was purified using the RNA Clean-up Kit (MACHEREY - NAGEL, Germany). Klenow enzyme labeling strategy was adopted after reverse transcription using reverse transcriptase. Labeled cDNA was purified with a PCR NucleoSpin Extract II Kit (MACHEREY - NAGEL, Germany) and re-suspended in elution buffer.
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| Channel 2 |
| Source name |
adjacent normal
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| Organism |
Homo sapiens |
| Characteristics |
tissue: cervical tumor
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| Extracted molecule |
total RNA |
| Extraction protocol |
UHRR was bought from Agilent,HBRR was bought from Ambion.tumor samples and normal tissues were collected from the Oncology Department of the Second Affiliated Hospital of Zhen Zhou University by related surgeons. Plasma samples were isolated by EDTA and centrifuged at 820 ×g for 10 min. The postoperative samples were collected 2 to 34 days after surgery.
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| Label |
Cy3
|
| Label protocol |
5ug isolated total RNA was treated with Rnase R (Epicentre, Madison, WI) to remove linear RNA. cDNA labeled with a fluorescent dye (Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction41, and the procedure has been improved by using CapitalBio circRNA Amplification and Labeling Kit (CapitalBio, Beijing) for producing higher yields of labeled cDNA. In detail, double-stranded cDNAs (containing the T7 RNA polymerase promoter sequence) were synthesized from 0.2 μg total RNA using the CbcScript reverse transcriptase with cDNA synthesis system according to the manufacturer’s protocol (Capitalbio, Beijing) with the T7 Oligo (dN). After completion of double-stranded cDNA (dsDNA) synthesis using DNA polymerase and RNase H, the dsDNA products about 12.5μL were subjected to 40μL in vitro transcription reactions at 37℃ for 14 h using a T7 Enzyme Mix. The amplified cRNA was purified using the RNA Clean-up Kit (MACHEREY - NAGEL, Germany). Klenow enzyme labeling strategy was adopted after reverse transcription using reverse transcriptase. Labeled cDNA was purified with a PCR NucleoSpin Extract II Kit (MACHEREY - NAGEL, Germany) and re-suspended in elution buffer.
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| Hybridization protocol |
27.5 μL of labeled samples were dissolved in 82.5 μL hybridization solution containing 3×SSC, 0.2% SDS, 5×Denhardt’s solution and 25% formamide. DNA in hybridization solution was denatured at 95℃ for 3 min prior to loading onto a the CapitalBio circRNA Human Gene Expression Microarray V1.0 (4x90K, CapitalBio, Beijing). Arrays hybridization were performed in a Agilent Hybridization Oven overnight at a rotation speed of 20 rpm and a temperature of 42℃ and washed with two consecutive solutions (0.2% SDS, 2× SSC at 42℃ for 5 min, and 0.2× SSC for 5 min at room temperature).
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| Scan protocol |
After washing the slides, the arrays were scanned by the Agilent Scanner G2565CA (Agilent, Santa Clara, CA). Acquired array images were analyzed by Agilent Feature Extraction (v10.7) software.
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| Description |
in the raw data file, rProcessedSignal represented Normal
in the raw data file, gProcessedSignal represented Tumor
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| Data processing |
Microarray data was extracted using Agilent Feature Extraction software. Data summarization, quantile normalization, quality control and most of the subsequent data processing were executed using GeneSpring software V12.0 (Agilent, Santa Clara, CA) or R software package.
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| Submission date |
Dec 01, 2016 |
| Last update date |
Mar 01, 2018 |
| Contact name |
Shasha Li |
| E-mail(s) |
[email protected]
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| Phone |
01062798728
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| Organization name |
University of Michigan
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| Street address |
1109 Maiden Ln, ct apt 104
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| City |
ANN ARBOR |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL22722 |
| Series (2) |
| GSE90737 |
Circular RNAs are super abundant in cervical tumor and plasma detected by high throughput microarray [cervical_cancer_circRNA] |
| GSE90741 |
Circular RNAs are super abundant in cervical tumor and plasma detected by high throughput microarray |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2411621_3T_3N_raw.txt.gz |
17.4 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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