|
| Status |
Public on Oct 30, 2010 |
| Title |
MGUS 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Bone marrow fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
disease state: MGUS
diagnostic test result: M component, IgGλ
cell type: bone marrow fibroblast
|
| Growth protocol |
Bone marrow fibroblasts are grown in 75 cm2 flask at 37°C (5% CO2) in DMEM containing 10% fetal calf serum (FCS), 100U/ml penicillin and streptomicin (Euroclone UK) until 80% confluence is reached for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted following the standard Trizol protocol (Invitrogen Life Technologies). RNA quantification and quality control was performed by Experion RNA STN-SENS Analysis on EXPERION automated electrophoresis station (Bio-Rad Laboratories).
|
| Label |
Cy5
|
| Label protocol |
1 μg aliquot of total RNA was retro-transcribed and labelled using the Amino Allyl MessageAmp® II aRNA Amplification Kit (Ambion) according to manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Bone marrow fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: pooled bone marrow fibroblasts
disease state: MGUS
|
| Growth protocol |
Bone marrow fibroblasts are grown in 75 cm2 flask at 37°C (5% CO2) in DMEM containing 10% fetal calf serum (FCS), 100U/ml penicillin and streptomicin (Euroclone UK) until 80% confluence is reached for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted following the standard Trizol protocol (Invitrogen Life Technologies). RNA quantification and quality control was performed by Experion RNA STN-SENS Analysis on EXPERION automated electrophoresis station (Bio-Rad Laboratories).
|
| Label |
Cy3
|
| Label protocol |
1 μg aliquot of total RNA was retro-transcribed and labelled using the Amino Allyl MessageAmp® II aRNA Amplification Kit (Ambion) according to manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Before hybridization, the Cy3 and Cy5 labeled samples were combined and dried in a speed vac. To the dried sample 330μl of hybridization buffer were added. The samples were denatured for 5 min at 65°C, snap cooled on ice for 1 min. The solution was pipetted onto the microarray slide placed in a hybridization chamber and the cover slip was placed carefully. Hybridization reaction was performed overnight in a sealed chamber (Corning® hybridization chambers, Sigma) at 42°C in a high-precision Techne Hybridizer Oven HB-1D (Barloworld Scientific Techne). Pre and post-hybridization washing was performed according to the protocol described in: Molecular Cloning a Laboratory Manual: Sambrook, Fritsch, Maniatis; Second edition, chapter 9, pag: 9.47-9.55
|
| Scan protocol |
Fluorescence signals were detected by analyzing the microarrays in a VersArray ChipReader® 5μm dual confocal laser scanner, with VersArray ChipReader v3.1 software (Bio-Rad Life Sciences Division); For each microarray slide, two images were produced by illuminating the array at 635 nm (excitation of Cy5) and 532 nm (Cy3). For both illuminations, photomultiplier tube (gain and light amplification) settings were at 1000 and laser power was set at 50%. All images were captured in TIFF format.
|
| Description |
ch1: Male; MGUS; M component, IgGλ
ch2: RNA of primary colture of bone marrow fibroblasts isolated from patients with monoclonal gammopathy of undetermined significance (MGUS).Total RNA were extrated from each primary coltures and 1 μg aliquot of total RNA from each samples were pooled to obtained a reference sample.
|
| Data processing |
Raw images were analyzed with VersArray Analyzer Software v4.5 (Bio-Rad Laboratories) using media pixel intensities for each spot. Global background was subtracted by biquadratic polynomial approximation, and cross-channel normalization was performed by local regression (LOESS); logarithmic transformation was then performed for each expression level.
|
| |
|
| Submission date |
Oct 28, 2010 |
| Last update date |
Oct 29, 2010 |
| Contact name |
angelina boccarelli |
| E-mail(s) |
angelina.boccarelli@uniba.it
|
| Phone |
390805478461
|
| Organization name |
University of Bari
|
| Department |
Department of Biomedical Sciences and Human Oncology
|
| Lab |
Experimental Chemotherapy
|
| Street address |
P.zza G. Cesare, 11
|
| City |
Bari |
| State/province |
Bari |
| ZIP/Postal code |
70124 |
| Country |
Italy |
| |
|
| Platform ID |
GPL2136 |
| Series (1) |
| GSE24990 |
Gene expression of bone marrow fibroblasts from monoclonal gammopathy of undetermined significance and active multiple myeloma |
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