|
| Status |
Public on Aug 01, 2014 |
| Title |
Rat1 cells 50 min PE Treatment |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rat-1 Fibroblast Serum PE 50 min
|
| Organism |
Rattus rattus |
| Characteristics |
cell line: derived embyonic fibroblast from Fisher 2408 Rat
cell type: fibroblast
treatment: phenylephrine
time: 50 min
|
| Treatment protocol |
Cells grown as described were stimulated by addition of filter sterilized phenylephrine (PE) stock in water to a final concentration of 10 uM, followed by gentle circular rotation to ensure mixing with minimal agitation. At the appropriate time the media was removed quickly followed by immediate addition of Trizol for total RNA extraction.
|
| Growth protocol |
A characterized rat-1 fibroblast cell line stably expressing HA-tagged human alpha-1a Adrenergic Receptor at 1.77±0.24 pmole/mg total protein was grown under 5% CO2 at 37°C in growth medium containing Dulbecco's Eagle Modified Medium supplemented with 10% fetal bovine serum (Hyclone, SH30071.03HI), penicillin (100 U/ml), streptomycin (100 µg/ml) and 0.5 mg/ml G418 to maintain selection. Two days prior to use (42-50h), cells at 75% confluence were trypsinized and plated at 2 to 2.5 million cells per 15 cm dish in growth medium without G418 and grown to about 75% confluence in growth medium under 5% CO2 at 37°C until treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following treatment, media was removed first by pouring then by aspiration for 5 secs followed by immediate addition of 10 ml of Trizol for total RNA extraction. Cells were then incubated in Trizol with rotation for 20 to 40 minutes at room temperature prior to storage at -80°C. Later, 5 ml of these Trizol preparations in 15 ml tubes were thawed and the total RNA extracted as recommended by the manufacturer employing the tabletop centrifuge modification. Total RNA was resuspended to 1ug/ul in water.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was reverse transcribed using Super script II and Oligo dT primers. RNA from individual time points was labeled with Cy5 while reference RNA was labeled with Cy3. In order to ensure all upregulated points were captured by the reference RNA, it was created by combining an equal amounts of RNA from untreated cells with a mix form across the time series (30 min, 1h, 2h, 8h and 24h). Details of labeling protocols can be found at http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/
|
| |
|
| Channel 2 |
| Source name |
Rat-1 Fibroblast Combined Reference
|
| Organism |
Rattus rattus |
| Characteristics |
cell line: derived embyonic fibroblast from Fisher 2408 Rat
cell type: fibroblast
|
| Treatment protocol |
Cells grown as described were stimulated by addition of filter sterilized phenylephrine (PE) stock in water to a final concentration of 10 uM, followed by gentle circular rotation to ensure mixing with minimal agitation. At the appropriate time the media was removed quickly followed by immediate addition of Trizol for total RNA extraction.
|
| Growth protocol |
A characterized rat-1 fibroblast cell line stably expressing HA-tagged human alpha-1a Adrenergic Receptor at 1.77±0.24 pmole/mg total protein was grown under 5% CO2 at 37°C in growth medium containing Dulbecco's Eagle Modified Medium supplemented with 10% fetal bovine serum (Hyclone, SH30071.03HI), penicillin (100 U/ml), streptomycin (100 µg/ml) and 0.5 mg/ml G418 to maintain selection. Two days prior to use (42-50h), cells at 75% confluence were trypsinized and plated at 2 to 2.5 million cells per 15 cm dish in growth medium without G418 and grown to about 75% confluence in growth medium under 5% CO2 at 37°C until treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following treatment, media was removed first by pouring then by aspiration for 5 secs followed by immediate addition of 10 ml of Trizol for total RNA extraction. Cells were then incubated in Trizol with rotation for 20 to 40 minutes at room temperature prior to storage at -80°C. Later, 5 ml of these Trizol preparations in 15 ml tubes were thawed and the total RNA extracted as recommended by the manufacturer employing the tabletop centrifuge modification. Total RNA was resuspended to 1ug/ul in water.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was reverse transcribed using Super script II and Oligo dT primers. RNA from individual time points was labeled with Cy5 while reference RNA was labeled with Cy3. In order to ensure all upregulated points were captured by the reference RNA, it was created by combining an equal amounts of RNA from untreated cells with a mix form across the time series (30 min, 1h, 2h, 8h and 24h). Details of labeling protocols can be found at http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/
|
| |
|
| |
| Hybridization protocol |
Cy5-labeled time point samples and an equimolar amount of rat reference RNA labeled with Cy3 were hybridized to a DNA chip containing the version 3 rat microarray (Operon Biotechnologies). After hybridization, slides were washed sequentially. Details of hybridization and washing protocols can be found at http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/
|
| Scan protocol |
Scanned on a Gene Pix 5000 scanner in the Duke University Microarray Core facility.
|
| Description |
40 min PE (10 uM) treated Rat-1 fibroblasts, growth conditions, harvested at 75% confluence.
|
| Data processing |
Data was analyzed using GeneSpring. Two color data was subject to Lowess normalization and background subtraction. Ratio of sample/reference (Cy5/Cy3) signal for each time point was then divided by the averaged sample/reference (Cy5/Cy3) signal observed for the three untreated replicates in order to normalize the data series to zero time RNA levels. All time point sample values were retained to ensure that elimination of very low mRNA levels in untreated samples did not remove higher activated expression levels.
Following Lowess normalization and background subtraction, the ratio of sample/reference (Cy5/Cy3) signal for each time point was divided by the average sample/reference (Cy5/Cy3) signal observed for the three untreated replicates in order to normalize the data series to zero-time, untreated RNA levels. This normalized ratio, representing the fold-change in poly-A mRNA relative to untreated cells, was subject to Log 10 transformation to generate the reported values.
Non-numeric values indicate signal is absent in this point but present at other time points.
Log 10 transformation of the normalized poly-A mRNA levels at each time point relative to the average of the triplet untreated zero points. Non-numeric values indicate signal is absent in this point but present at other time points.
Cy3 Reference counts following lowess normalization and background subtraction. Non-numeric values indicate signal is absent in this point but present at other time points.
Cy5 Sample counts following lowess normalization and background subtraction. Non-numeric values indicate signal is absent in this point but present at other time points.
|
| |
|
| Submission date |
Jul 31, 2014 |
| Last update date |
Aug 01, 2014 |
| Contact name |
Daniel P Morris |
| E-mail(s) |
dpmorris@llu.edu
|
| Organization name |
Loma LInda University
|
| Department |
Center for Perinatal Biology
|
| Lab |
Center for Perinatal Biology, rm A572
|
| Street address |
Loma LInda University
|
| City |
Loma Linda |
| State/province |
CA |
| ZIP/Postal code |
92350 |
| Country |
USA |
| |
|
| Platform ID |
GPL9207 |
| Series (1) |
| GSE59955 |
Time Course of Gene Expression in Rat-1 Fibroblasts Following Alpha-1a Adrenergic Receptor Stimulation with Phenylephrine. |
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