|
| Status |
Public on Sep 21, 2010 |
| Title |
12615-12617-Cy5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
culture type: control
|
| Organism |
Homo sapiens |
| Characteristics |
sample_name: 12615-12616
sex: M
sample_type: control
dkfz_patient_comments: B-CLL 45
culture_type: 0
time point (days): 0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation by Trizol extraction and purification on RNeasy Mini spin columns. Amplification of RNA by first- and second-strand cDNA synthesis. In vitro transcription of extracted cDNA by RiboMAX Large Scale RNA Production System T7.
|
| Label |
Cy3
|
| Label protocol |
Sample labelling by Klenow reaction using Cy3- or Cy5-labeld dUTP. Purification of labelled samples on Microcon YM-30 filter columns. Addition of blocking reagents: 25 μg Cot-1 DNA, 25 μg poly-A RNA, and 75 μg yeast tRNA.
|
| |
|
| Channel 2 |
| Source name |
culture_type: CM
|
| Organism |
Homo sapiens |
| Characteristics |
sample_name: 12615-12617
sex: M
sample_type: sample
dkfz_patient_comments: B-CLL 45
culture_type: CM
time point (days): 2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation by Trizol extraction and purification on RNeasy Mini spin columns. Amplification of RNA by first- and second-strand cDNA synthesis. In vitro transcription of extracted cDNA by RiboMAX Large Scale RNA Production System T7.
|
| Label |
Cy5
|
| Label protocol |
Sample labelling by Klenow reaction using Cy3- or Cy5-labeld dUTP. Purification of labelled samples on Microcon YM-30 filter columns. Addition of blocking reagents: 25 μg Cot-1 DNA, 25 μg poly-A RNA, and 75 μg yeast tRNA.
|
| |
|
| |
| Hybridization protocol |
Array platform: 70 mer oligonucleotides (Human Oligo Set 4.0; Operon, Cologne, Germany). Pretreatment of slides: 2 min in 0.2perc SDS; 2 min in ddH2O at RT; 2 min in ddH2O at 95C, followed by 1 min centrifugation at 1000 rpm. Application of preheated (60 min 60C, 10 min 70C), dye-labeled cDNA in Ultra-Hyb hybridization buffer on microarrays in a GeneTAC Hybridization Station. Hybridization for 40h at 42C with gentle agitation. Wash steps: 36C; 5 min 0.5xSSC, 0.1perc SDS; 3 min 0.05xSSC, 0.1perc SDS; 20s 0.05xSSC; 20s 0.05xSSC, 0.1perc Tween20.
|
| Scan protocol |
Scanning of hybridized microarrays at 5 μm resolution and variable PMT voltage to obtain maximal signal intensities with < 0.1perc probe saturation, a count ratio of 0.8-1.2 (Cy5/Cy3) and maximal congruence of histogram curves using a GenePix 4000B microarray scanner (Axon Instruments, Union City, USA).
|
| Description |
log2 ratio Cy5/Cy3
|
| Data processing |
Spots not recognized by the image analysis software, or not passing the quality criteria were excluded from the calculations. Raw fluorescence intensities were normalized applying the variance stabilization method (W. Huber et al., Bioinformatics 18 Suppl. 1, 2002).
|
| |
|
| Submission date |
Mar 26, 2010 |
| Last update date |
Apr 30, 2013 |
| Contact name |
Grischa Toedt |
| Organization name |
German Cancer Research Center (DKFZ)
|
| Department |
Molecular Genetics (B060)
|
| Lab |
Prof. Peter Lichter
|
| Street address |
Im Neuenheimer Feld 280
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9244 |
| Series (1) |
| GSE18192 |
Genome-Wide Expression Profiling of cultured B-CLL cells |
|
