|
| Status |
Public on Mar 01, 2010 |
| Title |
CLL CD14+ FOA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
UHR
|
| Organism |
Homo sapiens |
| Characteristics |
Universal Human Reference RNA (UHR) (Stratagene, Cedar Creek, TX), consisted of equal amount of total RNA from 10 human cancer cell lines, was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Agilent Technologies) in each experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
COMMERCIAL RNA (STRATAGENE)
|
| Label |
CY3
|
| Label protocol |
Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA)
|
| |
|
| Channel 2 |
| Source name |
CLL CD14+ FOA
|
| Organism |
Homo sapiens |
| Characteristics |
CD14+ CELLS PURIFIED FROM PERIPHERAL BLOOD OF A CLL PATIENT
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Mini Kit (QIAGEN, Valencia, CA, USA)
|
| Label |
CY5
|
| Label protocol |
Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA)
|
| |
|
| |
| Hybridization protocol |
825 ng of Cy5-labelled cRNA were mixed with the same amount of Cy3-labeled reference cRNA and then cRNA mixtures were fragmented to an average size of 50-100 nt by incubation at 60°C for 30 min using in situ Hybridization kit-plus (Agilent Technologies). Samples were hybridized for 17 h at 65°C on 4X44K Whole Human Genome Microarray (Agilent Technologies), comprising over 33’000 (60-mer) experimentally validated oligonucleotide probes and then scanned using a confocal laser scanner (Agilent Technologies).
|
| Scan protocol |
Fluorescence data were analyzed with Feature Extraction Software v.9.1 (Agilent Technologies).
|
| Description |
circulating endothelial cells (CEC), chronic lymphocytic leukemia (CLL),
|
| Data processing |
Result data from each scan (Log10 Cy5/Cy3) were imported into the gene expression analysis software Luminator (Rosetta Bio software, Seattle, WA, USA). Two-dimensional clustering analysis was performed using an agglomerative algorithm with an average link heuristics and a correlation with mean subtraction.The identification of genes differentially expressed between subgroups was performed using both Significant Analysis of Microarray (SAM) algorithm with false discovery ratio (FDR)<5% and enhanced Analysis of Variance (ANOVA) with p-value <0.001. To increase the statistical power, the enhanced ANOVA uses as input data both expression level and the estimated technology error associated with the expression level. As a result, the false-positive rate is reduced when the number of replicates is small and, then, sensitivitydetection is increased.
|
| |
|
| Submission date |
Feb 16, 2009 |
| Last update date |
Dec 28, 2009 |
| Contact name |
Rossana Maffei |
| E-mail(s) |
rossana.maffei@unimore.it
|
| Phone |
+39 059 4222715
|
| Organization name |
University of Modena and Reggio Emilia
|
| Department |
Dept of Hematology and Oncology
|
| Lab |
Lab of Molecular Hematology
|
| Street address |
Via del Pozzo 71
|
| City |
Modena |
| State/province |
Modena |
| ZIP/Postal code |
41100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE14853 |
Circulating endothelial cells in patients with chronic lymphocytic leukemia: clinical and biological characterization |
|
