|
| Status |
Public on Dec 31, 2021 |
| Title |
Chip No: 288pB from patient 3326 |
| Sample type |
RNA |
| |
|
| Source name |
peripheral whole blood sample after 6 weeks of treatment
|
| Organism |
Homo sapiens |
| Characteristics |
molecular response: MMR
gender: male
pretreatment hydroxyurea: yes
pretreatment interferon: yes
pretreatment busulfan: yes
pretreatment others: no
dose: Imatinib 400 mg/day
time point: 2
time: 6 wk
subject: patient 3326
tissue: peripheral blood
treatment: imatinib
disease state: chronic myeloid leukemia
|
| Treatment protocol |
primary peripheral whole blood samples prior treatment (n=135), subset after 6 weeks of treatment (n=65) either after 400mg imatinib once daily or 800mg imatinib once daily
|
| Growth protocol |
not applicable,no cell culture involved
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral whole blood samples, preserved in PAXgene tubes containing an RNA stabilizing agent, stored at or below -20°C , processing was performed using the Affymetrix Blood RNA concentration kit according to the manufacturers' instructions. In brief, blood cells were lyzed sedimented and pelleted, Following homogenization by centrifugation through shredder columns the supernatant was transferred, centrifuged and RNA bound to column membranesDNA was digested by DNAse I treatment prior to concentration using the Affymetrix Blood RNA concentration kit. Total RNA was consecutively depleted of haemoglobin mRNA applying the Globin Reduction kit following the manufacturer’s protocol (Preanalytix, Switzerland).
|
| Label |
biotin
|
| Label protocol |
Using One Cycle cDNA Synthesis- and One Cycle Target Labelling-Kit from Affymetrix biotinylated hybridization targets were generated from 1.5 µg total RNA. Purified oligo-dT-T7-primed double-stranded cDNA was amplified by T7 polymerase in single round cRNA amplification step. Incorporation of a modified nucleotide during the latter step allowed subsequent biotinylation. Purified cRNA was fragmented and 20 µg were used for GeneChip hybridization following the manufacturer’s protocols.
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| |
|
| Hybridization protocol |
Purified cRNA was fragmented and 20 µg were used for GeneChip hybridization following the manafacturer’s protocols (Affymetrix GeneChip Expression Analysis Technical Manual Revison #4; Eukaryotic Target Preparation, Revison #3). After staining and washing of the hybridized arrays in an Affymetrix fluidic station 450 following the EukGE-ws2v4 protocol.
|
| Scan protocol |
Fluorescence intensity signals were read by an Affymetrix 3000 scanner
|
| Data processing |
Image analysis was performed with the GCOS software (version 1.2) and for subsequent bioinformatics analysis the statistical programming language “R” (V2.3) and additional software package from the BioConductor library (Release 2.6) were used. Briefly, for identifying genes predicting imatinib response in this group of pre-treated CML patients we followed a main workflow consisting of filtering genes by variance of expression, consecutive identification of differentially expressed genes and eventually applying linear discriminatory analysis for response prediction. This approach was run in a nested-cross validation manner, in which the full data set was at random divided into test and training set and this division was repeated 40 times. The resulting training sets were again split into 40 randomly chosen training and test-subsets. Within each of these training subsets feature selection and all pre-processing steps (filtering, differential expression) were performed. Features were selected for testing in the prediction algorithm based on their differential expression calculated by t-test and adjusted for multiple testing using Benjamini-Hochberg correction, if they showed a at least 1.5-fold difference in expression levels and had a adjusted significance <0.01%.
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| |
|
| Submission date |
Feb 22, 2013 |
| Last update date |
Dec 31, 2021 |
| Contact name |
Stefan Schmidt |
| E-mail(s) |
stefan.schmidt@i-med.ac.at
|
| Organization name |
Medical University Innsbruck
|
| Street address |
Anichstr. 35
|
| City |
Innsbruck |
| ZIP/Postal code |
6020 |
| Country |
Austria |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE44589 |
Gene expression-based imatinib response prediction in chronic phase CML |
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