|
| Status |
Public on Mar 22, 2006 |
| Title |
TG+1C-3 vs NTG1C-5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cultured murine ASM (airway smooth muscle) cells from tracheal explants of NTG mice
|
| Organism |
Mus musculus |
| Characteristics |
murine ASM cells of NTG mice
|
| Growth protocol |
Primary cultures of murine ASM cells were established from tracheal explants of NTG and 2AR-OE mice. Briefly, the trachea between the larynx and main stem bronchi was removed and placed in a sterile Petri dish containing Hanks' balanced saline solution supplemented with a 2x concentration of antibiotic-antimycotic solution. After additional surrounding tissue was removed with the aid of a dissecting microscope, the tracheal segment was split longitudinally and dissected into 2-3 mm squares. All of the segments from a single trachea were then placed intima side down in a sterile 60-mm dish. After allowing the explants to adhere, 2.5 ml of Dulbecco's modified Eagle's medium supplemented with 20% FCS and 2x antibiotic-antimycotic was added to cover the explants. Explanted trachea were subsequently removed when there was local confluency. Once the initial seed dish became confluent, cells were harvested by trypsinization and passed into 75-cm2 flasks. As previously described {}, >90% of these cells were smooth muscle cells, as determined by immunohistochemistry performed with an antibody raised against smooth muscle -actin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cells between passages 8 and 10 using TRI Reagent (Molecular Research Center, Cincinnati, OH), and resuspended in DEPC-treated water. Each sample was visually inspected for degradation in agarose gels stained with ethidium bromide, and their quality was further checked for RNase degradation using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy5
|
| Label protocol |
The RNA samples are converted to cDNA using a SuperScript III based reverse transcription. Then the cDNA samples are labeled with Cy3 or Cy5 dyes and hybridized to pre-printed, pre-hybridized amine coated slides where a competitive hybridization occurs.
|
| |
|
| Channel 2 |
| Source name |
cultured murine ASM (airway smooth muscle) cells of 2AR-OE mice
|
| Organism |
Mus musculus |
| Characteristics |
murine ASM cells of 2AR-OE mice
|
| Growth protocol |
Primary cultures of murine ASM cells were established from tracheal explants of NTG and 2AR-OE mice. Briefly, the trachea between the larynx and main stem bronchi was removed and placed in a sterile Petri dish containing Hanks' balanced saline solution supplemented with a 2x concentration of antibiotic-antimycotic solution. After additional surrounding tissue was removed with the aid of a dissecting microscope, the tracheal segment was split longitudinally and dissected into 2-3 mm squares. All of the segments from a single trachea were then placed intima side down in a sterile 60-mm dish. After allowing the explants to adhere, 2.5 ml of Dulbecco's modified Eagle's medium supplemented with 20% FCS and 2x antibiotic-antimycotic was added to cover the explants. Explanted trachea were subsequently removed when there was local confluency. Once the initial seed dish became confluent, cells were harvested by trypsinization and passed into 75-cm2 flasks. As previously described {}, >90% of these cells were smooth muscle cells, as determined by immunohistochemistry performed with an antibody raised against smooth muscle -actin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cells between passages 8 and 10 using TRI Reagent (Molecular Research Center, Cincinnati, OH), and resuspended in DEPC-treated water. Each sample was visually inspected for degradation in agarose gels stained with ethidium bromide, and their quality was further checked for RNase degradation using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
The RNA samples are converted to cDNA using a SuperScript III based reverse transcription. Then the cDNA samples are labeled with Cy3 or Cy5 dyes and hybridized to pre-printed, pre-hybridized amine coated slides where a competitive hybridization occurs.
|
| |
|
| |
| Scan protocol |
Fluorescence intensity analyses and background subtraction were performed using an Axon Instruments scanner and GenePix software
|
| Description |
murine ASM cells
|
| Data processing |
Lowess normalization and per-chip normalization using GeneSpring GX 7.3
|
| |
|
| Submission date |
Mar 20, 2006 |
| Last update date |
Mar 21, 2006 |
| Contact name |
Bruce J Aronow |
| E-mail(s) |
bruce.aronow@chmcc.org
|
| Phone |
513-636-4865
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Street address |
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL1406 |
| Series (1) |
| GSE4499 |
response to persistent beta 2-adrenergic receptor signaling |
|
