|
| Status |
Public on May 18, 2016 |
| Title |
ascending colon cancer_with liver metastasis_original [Exp 5] |
| Sample type |
RNA |
| |
|
| Source name |
ascending colon cancer_with liver metastasis
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: colorectal cancer (CRC) patient with liver metastasis
age (yrs): 64
gender: M
tissue: peripheral blood
|
| Treatment protocol |
Freshly drawn peripheral blood from healthy volunteers was irradiated, then diluted 1:1 with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated for 6 or 24 hours at 37C in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Versagene Blood RNA Purification kit (Gentra Systems, Minneapolis MN) following the manufacturer's recommendations. The protocol includes differential lysis of red and white blood cells, and an on-column DNase digestion. Globin message was further reduced using GLOBINclear (Ambion Inc., Austin, TX) to specifically remove both a- and b- globin. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
lncRNAs and mRNAs expression in the colon cancer tissue of CRC patients with liver metastasis
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Nov 16, 2015 |
| Last update date |
May 18, 2016 |
| Contact name |
Dong Chen |
| E-mail(s) |
[email protected]
|
| Organization name |
First Affiliated Hospital, Zhejiang University
|
| Street address |
79# Qingchun Rd
|
| City |
Hangzhou |
| ZIP/Postal code |
310003 |
| Country |
China |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE75050 |
Genome-wide analysis of long noncoding RNA (lncRNA) expression in colorectal cancer tissues from patients with liver metastasis |
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