|
| Status |
Public on May 26, 2009 |
| Title |
3T3-L1 day 1 |
| Sample type |
other |
| |
|
| Channel 1 |
| Source name |
3T3-L1 cells prior to induction of adipocyte differentiation (day 0)
|
| Organism |
Mus musculus |
| Characteristics |
agent: untreated (control)
|
| Treatment protocol |
For DMI induction, 1mM Dexamethasone, 0.5mM IBMX, and 5mg/ml Insulin were added to culture medium two days after the cells reached confluence. After 48 hours, the culture medium was replaced with DMEM containing 10% FBS and 5mg/ml Insulin. The same medium was replaced every 48 hours until pre-adipocytes differentiated into mature adipocytes.
|
| Growth protocol |
3T3-L1 cells were cultured in a 5% CO2 humidified atmosphere in Dulbecco's Modified Eagle's Medium (DMEM)/high glucose with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, and 100mg/ml streptomycin.
|
| Extracted molecule |
other |
| Extraction protocol |
The standard phenol/chloroform extraction protocol was used to isolate total RNA. MicroRNAs were isolated from total RNA using a FlashPAGE fractionator (Ambion, TX). 100mg of total RNA was loaded onto FlashPAGE precast gels and small size RNAs collected after electrophoresis at 80V for 12 minutes. Small size RNA was purified using the FlashPAGE Reaction Clean Up Kit (Ambion, TX).
|
| Label |
Cy3
|
| Label protocol |
miRNA labeling was carried out according to directions from the Ambion mirVana™ miRNA Labeling Kit. Briefly, 1mg of extracted miRNA was appended with amine-NTPs at 37 degrees celcius for 2 hrs. Samples were washed to remove free NTPs. Cy3 and Cy5-NHS ester dye was coupled to amine modified miRNA at room temperature for 1hr in the dark. Samples were washed to remove free dye. Labeled samples were imediately used for array hybridization. The common day 0 reference was labeled with Cy3, and days 1, 4, and 7 were labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
3T3-L1 cells, 1 day following induction of adipocyte differentiation with DMI cocktail
|
| Organism |
Mus musculus |
| Characteristics |
agent: DMI cocktail (1mM Dexamethasone, 0.5mM IBMX, and 5mg/ml Insulin)
time: 1 day
|
| Treatment protocol |
For DMI induction, 1mM Dexamethasone, 0.5mM IBMX, and 5mg/ml Insulin were added to culture medium two days after the cells reached confluence. After 48 hours, the culture medium was replaced with DMEM containing 10% FBS and 5mg/ml Insulin. The same medium was replaced every 48 hours until pre-adipocytes differentiated into mature adipocytes.
|
| Growth protocol |
3T3-L1 cells were cultured in a 5% CO2 humidified atmosphere in Dulbecco's Modified Eagle's Medium (DMEM)/high glucose with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, and 100mg/ml streptomycin.
|
| Extracted molecule |
other |
| Extraction protocol |
The standard phenol/chloroform extraction protocol was used to isolate total RNA. MicroRNAs were isolated from total RNA using a FlashPAGE fractionator (Ambion, TX). 100mg of total RNA was loaded onto FlashPAGE precast gels and small size RNAs collected after electrophoresis at 80V for 12 minutes. Small size RNA was purified using the FlashPAGE Reaction Clean Up Kit (Ambion, TX).
|
| Label |
Cy5
|
| Label protocol |
miRNA labeling was carried out according to directions from the Ambion mirVana™ miRNA Labeling Kit. Briefly, 1mg of extracted miRNA was appended with amine-NTPs at 37 degrees celcius for 2 hrs. Samples were washed to remove free NTPs. Cy3 and Cy5-NHS ester dye was coupled to amine modified miRNA at room temperature for 1hr in the dark. Samples were washed to remove free dye. Labeled samples were imediately used for array hybridization. The common day 0 reference was labeled with Cy3, and days 1, 4, and 7 were labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
labeled miRNA was hybridized to custom arrays at 42 degrees celcius for 12h.
|
| Scan protocol |
images were scaned according to standard UTSW microarray core facility protocols.
|
| Description |
total miRNA profiling by array
|
| Data processing |
Scanned images were processed using GenePix software.
The normalized signal represents the data after Lowess normalization, ratio computation (Cy5/Cy3), and log2 transformation of raw data
The term raw signal values refer to the linear data after thresholding to 1.0 and summarization for the individual channels (Cy3 and Cy5). Summarization was performed by computing the geometric mean of replicate spots.
|
| |
|
| Submission date |
May 26, 2009 |
| Last update date |
May 26, 2009 |
| Contact name |
Scott Andrew Ochsner |
| E-mail(s) |
sochsner@bcm.edu
|
| Phone |
713-798-6227
|
| Organization name |
Baylor College of Medicine
|
| Department |
Molecular and Cellular Biology
|
| Lab |
SPP: Signaling Pathways Project
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL8594 |
| Series (1) |
| GSE16229 |
Detection of microRNA expression during 3T3-L1 pre-adipocyte differentiation using a custom made microarray chip. |
|
