|
| Status |
Public on Apr 02, 2012 |
| Title |
MDEbiorep2techrep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RD cells transduced with empty vector pCLBabe
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: RD cells
treatment: transduced with empty vector pCLBabe
|
| Treatment protocol |
RD cells were transduced with either MD~E or empty vector pCLBabe retroviruses, selected with puromycin for 48 hours and differentiated for 24 hours with selection
|
| Growth protocol |
RD cells were obtained from ATCC (American Type Culture Collection) in approximately 1990, and all analyses have been performed on cells that originated from low passage number frozen aliquots. Cells were maintained in DMEM with 10% bovine calf serum and 1% Pen-Strep (Gibco). Low-serum differentiation media consisted of DMEM with 1% horse serum, 1% Pen-Strep and 10 ug/mL insulin and transferrin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol and acid-phenol purification
|
| Label |
Cy3,Cy5
|
| Label protocol |
miRNAs were labeled using Exiqon’s miRCURY LNA labeling kit
|
| |
|
| Channel 2 |
| Source name |
RD cells transduced with MD~E forced dimer
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: RD cells
treatment: transduced with MD~E forced dimer
|
| Treatment protocol |
RD cells were transduced with either MD~E or empty vector pCLBabe retroviruses, selected with puromycin for 48 hours and differentiated for 24 hours with selection
|
| Growth protocol |
RD cells were obtained from ATCC (American Type Culture Collection) in approximately 1990, and all analyses have been performed on cells that originated from low passage number frozen aliquots. Cells were maintained in DMEM with 10% bovine calf serum and 1% Pen-Strep (Gibco). Low-serum differentiation media consisted of DMEM with 1% horse serum, 1% Pen-Strep and 10 ug/mL insulin and transferrin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol and acid-phenol purification
|
| Label |
Cy5,Cy3
|
| Label protocol |
miRNAs were labeled using Exiqon’s miRCURY LNA labeling kit
|
| |
|
| |
| Hybridization protocol |
Pre-hybridized arrays were treated following Schott’s protocol for the Nexterion’s SLIDE-E surface. Experimental and reference samples were combined and resuspended in 200 μl of 1x miRCURY hybridization buffer (Exiqon, Inc., Woburn, MA), incubated for 5 minutes at 95°C, and subsequently centrifuged for 5 minutes at 14,000xg. Combined sample pairs were co-hybridized to each array using the SureHyb Microarray Hybridization System (Agilent Technologies , Santa Clara, CA). Hybridization chambers were rotated at 4 rpm at 60°C for 16-18 hours using a Robbins Scientific 400 Hybridization Incubator (SciGene Corporation, Sunnyville, CA). Post-hybridized arrays were washed for 2 minutes in 2XSSC + 0.2% SDS at 60°C, followed by 2x washes in 1xSSC at RT for 2 min each, and finishing with a final wash in 0.2XSSC at RT for 2 min. Slides were dried by centrifugation at 200xg for 5 min.
|
| Scan protocol |
Slides were scanned at 10um resolution using a GenePix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA). Image quantification was performed using GenePix Pro 6.0 Microarray Image Analysis software (Molecular Devices Corporation, Sunnyvale, CA).
|
| Data processing |
For each array, we took log2 ratios of the green and red signals at each spot. As a form of normalization, we calculated the mean ratio for four probes representing U6 snRNA, which should not change on MD~E introduction, and subtracted the mean U6 log2 ratio from all other log ratios on the same array. We then averaged the log ratios between dye-swap replicates. Signals on these arrays were generally weak and somewhat inconsistent across dye-swaps and replicates, making it very difficult to use more sophisticated normalization methods. We used these arrays only to choose the most obviously changed miRNAs: any findings important to our publication were verified independently using miRNA Northern blots. These array results are not of high enough quality to make general statements about expression changes across the full repertoire of human miRNAs.
|
| |
|
| Submission date |
Feb 07, 2012 |
| Last update date |
Apr 03, 2012 |
| Contact name |
Janet M. Young |
| E-mail(s) |
jayoung@fhcrc.org
|
| Phone |
206 667 1471
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Department |
Division of Human Biology
|
| Lab |
Tapscott
|
| Street address |
1100 Fairview Avenue N., C3-168, P.O. Box 19024
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL6844 |
| Series (2) |
| GSE35606 |
miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells (miRNA) |
| GSE35921 |
miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells |
|
