|
| Status |
Public on Jan 01, 2009 |
| Title |
Dendritic Cells vs. Peripheral Blood Monocyctes |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Dendritic Cells
|
| Organism |
Mus musculus |
| Characteristics |
wild-type C57BL/6N mice
gender: male and female
age: six to nine weeks
cells isolated by FACS
|
| Biomaterial provider |
Charles River (Sulzbach, Germany)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After sorting, cells were frozen at -80°C in RLT lysis buffer (Qiagen) with 1% beta-mercaptoethanol (Sigma). RNA from highly purified cell populations was isolated using an RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. Quantification and purity of RNA was determined with an Agilent Bioanalyzer 2100 (Agilent Biosystems). Only those RNA preparations exceeding absorbance ratios of A260/280 nm > 1.90 and of a total amount of RNA greater than 200 ng were used for microarray experiments.
|
| Label |
Cy3, Cy5
|
| Label protocol |
Labelling was performed according to the Two-Color Microarray-Based Gene Expression Analysis Protocol version 5.5 (Agilent) using an Agilent RNA Spike-In Kit, a Low RNA Input Linear Amp Kit and a Cyanine CTP2-color dye pack (Agilent Technologies, Wilmington, DE). Per reaction, 1 µg of total RNA was used. RNA was pooled using comparable amounts of total RNA (as assessed by Nanodrop analysis) from four animals.
|
| |
|
| Channel 2 |
| Source name |
Peripheral Blood Monocytes
|
| Organism |
Mus musculus |
| Characteristics |
wild-type C57BL/6N mice
gender: male and female
age: six to nine weeks
cells isolated by FACS
|
| Biomaterial provider |
Charles River (Sulzbach, Germany)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After sorting, cells were frozen at -80°C in RLT lysis buffer (Qiagen) with 1% beta-mercaptoethanol (Sigma). RNA from highly purified cell populations was isolated using an RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. Quantification and purity of RNA was determined with an Agilent Bioanalyzer 2100 (Agilent Biosystems). Only those RNA preparations exceeding absorbance ratios of A260/280 nm > 1.90 and of a total amount of RNA greater than 200 ng were used for microarray experiments.
|
| Label |
Cy5, Cy3
|
| Label protocol |
Labelling was performed according to the Two-Color Microarray-Based Gene Expression Analysis Protocol version 5.5 (Agilent) using an Agilent RNA Spike-In Kit, a Low RNA Input Linear Amp Kit and a Cyanine CTP2-color dye pack (Agilent Technologies, Wilmington, DE). Per reaction, 1 µg of total RNA was used. RNA was pooled using comparable amounts of total RNA (as assessed by Nanodrop analysis) from four animals.
|
| |
|
| |
| Hybridization protocol |
The labeled RNA pools were hybridized overnight at 60°C in Agilent in.situ hybridization buffer using Agilent Hybridization chambers.
|
| Scan protocol |
Slides were scanned using a GenePix 4100A Scanner (Axon Instruments, Downingtown, PA). PMT gains were adjusted to get a use the entire dynamic range of the scanner. Image analysis was performed with GenePix Pro 5.0 software.
|
| Description |
Trafficking of monocytes into lung tissue and their differentiation into lung resident macrophages and dendritic cells is supposed to be regulated by the expression of specific gene clusters, which promote cell-cell interaction, migration and matrix degradation and the acquisition of tissue specific cellular phenotypes. Traffic related gene clusters include chemokines, integrins, and tissue-degrading matrix metallopeptidases, for all of which members have been shown to be functionally important. A complete picture, however, of the gene clusters that are regulated during in vivo migration and differentiation of peripheral blood monocytes under non-inflammatory conditions has not been obtained yet. Currently, adaptive changes of cellular phenotypes cannot be directly assessed by cell fate mapping during the slow trafficking of mononuclear phagocytes to lung tissue under steady-state conditions. Therefore, as an alternative approach to gain a better insight into the genetic programs that drive the mononuclear phagocyte migration and differentiation processes, the transcriptomes of circulating monocytes were compared with their lung tissue mononuclear phagocyte progeny.
|
| Data processing |
Data were evaluated using the R software and the limma package from BioConductor. The spot intensities were corrected for local background using the method of Edwards with an offset of 64 to stabilize the variance of low-intensity spots. The M/A data were LOESS normalized before averaging. Genes were ranked for differential expression using a moderated t-statistic.
|
| |
|
| Submission date |
Nov 11, 2008 |
| Last update date |
Nov 14, 2008 |
| Contact name |
Jochen Wilhelm |
| Organization name |
Uniklinikum Giessen
|
| Department |
ECCPS Microarray Unit
|
| Street address |
Gaffkystrasse 10
|
| City |
Giessen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE13558 |
Lung Trafficking of Macrophages |
|
