|
| Status |
Public on Dec 22, 2011 |
| Title |
slowly proliferating single cell derived mesenchymal cell from bone marrow 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Human Control gDNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Promega gDNA female, G152A Lot. 25707301
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A maximum of 5x10^6 cells (RPCs, MPCs and SPCs at six weeks) were centrifuged for 5 minutes at 300 g. The pellet was resuspended in 200 μl PBS, and 20 μl proteinase K (Qiagen DNeasy Blood & Tissue Kit) and 4 μl RNase A (100 mg/ml) were added, mixed on a vortex mixer, and incubated for 2 min at room temperature. Then the suspension was spun in a microcentrifuge for 30 seconds at 6,000 g to drive the contents off the walls and lid. Genomic DNA (gDNA) was isolated using a Qiagen DNeasy Blood & Tissue Kit and Agilent’s recommended procedure (http://www.chem.agilent.com/Library/usermanuals/Public/G4410-90040 CGH Bravo Protocol 1.1.pdf). The final elution volume was 400 μl in Buffer AE. The yield and purity of the gDNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). To assess the quality and average molecular weight of the collected gDNA, aliquots from each sample were subjected to agarose gel electrophoresis. For experimentation, high-quality gDNA samples with a 260/280 ratio of 1.8 to 2.0 (the 260/230 ratio for pure DNA is >1.8) were used.
|
| Label |
Cy3
|
| Label protocol |
1.0 μg aliquots of sample DNA from RPCs, MPCs or SPCs, as well as reference DNA (Promega, female, p/n G1521), were digested with AluI and RsaI restriction enzymes and fluorescently labeled with Cy5 (test) or Cy3 (reference) using an Agilent Genomic DNA Enzymatic Labeling Kit (p/n 5190-0449).
|
| |
|
| Channel 2 |
| Source name |
Human Mesenchymal Stem Cells
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
age: 20 y
cell type: slowly proliferating single cell derived mesenchymal cell from bone marrow
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A maximum of 5x10^6 cells (RPCs, MPCs and SPCs at six weeks) were centrifuged for 5 minutes at 300 g. The pellet was resuspended in 200 μl PBS, and 20 μl proteinase K (Qiagen DNeasy Blood & Tissue Kit) and 4 μl RNase A (100 mg/ml) were added, mixed on a vortex mixer, and incubated for 2 min at room temperature. Then the suspension was spun in a microcentrifuge for 30 seconds at 6,000 g to drive the contents off the walls and lid. Genomic DNA (gDNA) was isolated using a Qiagen DNeasy Blood & Tissue Kit and Agilent’s recommended procedure (http://www.chem.agilent.com/Library/usermanuals/Public/G4410-90040 CGH Bravo Protocol 1.1.pdf). The final elution volume was 400 μl in Buffer AE. The yield and purity of the gDNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). To assess the quality and average molecular weight of the collected gDNA, aliquots from each sample were subjected to agarose gel electrophoresis. For experimentation, high-quality gDNA samples with a 260/280 ratio of 1.8 to 2.0 (the 260/230 ratio for pure DNA is >1.8) were used.
|
| Label |
Cy5
|
| Label protocol |
1.0 μg aliquots of sample DNA from RPCs, MPCs or SPCs, as well as reference DNA (Promega, female, p/n G1521), were digested with AluI and RsaI restriction enzymes and fluorescently labeled with Cy5 (test) or Cy3 (reference) using an Agilent Genomic DNA Enzymatic Labeling Kit (p/n 5190-0449).
|
| |
|
| |
| Hybridization protocol |
Experimental and reference targets for each hybridization were purified using a Microcon YM-30 column (Millipore) and validated using a NanoDrop ND-1000 to ensure the yield and specific incorporation of the labeled gDNA. Labeled test and reference DNAs were combined, denatured, pre-annealed with Cot-1 DNA (Invitrogen, Carlsbad, CA) and blocking reagent (Agilent), and then hybridized to Agilent Human 4x180K CGH arrays (Design ID = 022060) for 24 hours in a rotating oven (Agilent Technologies) at 65°C and 20 rpm.
|
| Scan protocol |
After the hybridization, the array slides were washed and then scanned at 3 μm resolution with an Agilent G2565CA Scanner. Data were extracted from the microarray images using Agilent Feature Extraction Software v10.5.1.1 with the Feature Extraction protocol CGH-105_Dec09. These ratio data, along with the associated error values and flagged features, were imported into the DNA Analytics Software v5.0.14 (Agilent).
|
| Data processing |
To make aberration calls, an aberration detection algorithm, ADM-2, was used with a threshold of 5.5 and an aberration filter set at 2 for the minimum number of probe regions and 0.25 for the minimum absolute average log2 ratio for regions in the DNA Analytics to reduce false positives.
|
| |
|
| Submission date |
Dec 15, 2011 |
| Last update date |
Dec 22, 2011 |
| Contact name |
Yo Mabuchi |
| E-mail(s) |
yomabuchi@2009.jukuin.keio.ac.jp
|
| Organization name |
Keio University School of Medicine
|
| Department |
Physiology
|
| Street address |
35 Shinanomachi, Shinjuku-ku
|
| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10123 |
| Series (1) |
| GSE34484 |
LNGFR- and Thy-1-based prospective isolation of human mesenchymal stem cells reveals functionally distinct subpopulations. |
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