|
| Status |
Public on Jun 05, 2012 |
| Title |
HCC derived γδT_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Human HCC-derived γδT cells, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Hepatocellular carcinoma
gender: male
|
| Treatment protocol |
Freshly acquired HCC tissue and paired peritumor liver tissue were minced into small pieces of ~1mm3, then digested in an enzyme cocktail (1mg/ml collagenase type IV, 0.02mg/ml DNase in HBSS with 10% FBS) for 1 hour at 37°C. After filtration and extensive wash, the cells were subjected to discontinous density gradient centrifugation in Percoll. After removal of dead cells and incubation with components of TCRγ/δ isolation kit (Mitenyi Biotec), γδ T cells were positively selected from the single-cell suspension using autoMACS Separator (Miltenyi Biotec) with recommended programs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted from freshly purified γδ T cells using Trizol reagent (Invitrogen) following the manufacturer's recommendation. Sample RNA Quality Control was performed with using Bioanalyzer 2100 to evaluate the RNA integrity from all samples. The NanoDrop ND-1000 was used for accurately measuring RNA concentrations (OD260), protein contamination (ratio of OD260/OD280) and organic compound contamination (ratio of OD260/OD230).
|
| 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 |
the arrays were scanned by the Agilent Scanner G2505B.
|
| Description |
Gene expression of HCC-derived γδT cells
|
| Data processing |
Agilent Feature Extraction software (version 10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies).
|
| |
|
| Submission date |
Jun 04, 2012 |
| Last update date |
Jun 05, 2012 |
| Contact name |
Yong Yi |
| E-mail(s) |
yiyong1984@gmail.com
|
| Phone |
15821328520
|
| Organization name |
Liver Cancer Institute
|
| Street address |
180 Fenglin Road
|
| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE38476 |
Investigation of the gene expression pattern of human hepatocellular carcinoma (HCC) infiltrating γδT cells |
|
