|
| Status |
Public on Oct 10, 2009 |
| Title |
T-47D |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
breast cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: T-47D
|
| Growth protocol |
Cells were grown in respective media to 70% confluence under standard growth conditions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Rneasy Kit (Qiagen) per manufactures instructions
|
| Label |
Cy5
|
| Label protocol |
750 ng of total RNA were primed with T7 primer at 65C for 10 min, then reverse transcribed at 42C for 2 hr in the presence of MMLV reverse transcriptase (15 units/ul) and and 50 uM each dATP, dGTP, dCTP and dTTP . After incubation, the reaction was heated at 65C for 15 min to inactivate the MMLV RT. Transcription buffer, DTT, NTP mix, PEG, RNaseOut, inorganic pyrophosphorylase, and T7 polymerase were added as per Agilent Low RNA Input Amplification Kit, along with 1.2 ul of 100nmol Cy5-CTP or Cy3-CTP. The transcription mix was incubated 40C for
|
| |
|
| Channel 2 |
| Source name |
pooled breast cell line reference
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: pooled breast cell lines
|
| Growth protocol |
Cells were grown in respective media to 70% confluence under standard growth conditions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Rneasy Kit (Qiagen) per manufactures instructions
|
| Label |
Cy3
|
| Label protocol |
750 ng of total RNA were primed with T7 primer at 65C for 10 min, then reverse transcribed at 42C for 2 hr in the presence of MMLV reverse transcriptase (15 units/ul) and and 50 uM each dATP, dGTP, dCTP and dTTP . After incubation, the reaction was heated at 65C for 15 min to inactivate the MMLV RT. Transcription buffer, DTT, NTP mix, PEG, RNaseOut, inorganic pyrophosphorylase, and T7 polymerase were added as per Agilent Low RNA Input Amplification Kit, along with 1.2 ul of 100nmol Cy5-CTP or Cy3-CTP. The transcription mix was incubated 40C for
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to Agilent Human version 2 microarray slides enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization at 64C for 17hr, slides were washed sequentially in SSPE solutions, dried and scanned.
|
| Scan protocol |
Scanned on an Agilent G2565BA scanner.
Images were quantified using Agilent Feature Extraction Software (version A.7.5.1.1).
|
| Description |
n/a
|
| Data processing |
Agilent Feature Extraction Software Version: 7.5
SpotFinder_Version: Version A.7.5.1 Jun 1 2004 09:41:20
CornerMethod: Auto Find Corners
CalcSpotStats_Version: Version A.7.5.1 Jun 1 2004 09:41:37
CalcSpotStats_SpotStatsMethod: Cookie Cutter Method
CalcSpotStats_RejectMethod: Interquartile Range Based
CalcSpotStats_CalculateSpotSize: Off
OutlierFlagger_Version: Version A.7.5.1 Jun 1 2004 09:44:12
OutlierFlagger_NonUnifOLOn: Nonuniformity Outlier Flag On
OutlierFlagger_PopnOLOn: Population Outlier Flagging turned On
BGSubtractor_Version: Version A.7.5.1 Jun 1 2004 09:42:46
BGSubtractor_BGSubMethod: Local background coresponding to each feature for background subtraction (local method)
BGSubtractor_BackgroundCorrectionOn: Globally adjust background turned off
BGSubtractor_SpatialDetrendOn: Spatial detrend turned off
DyeNorm_Version: Version A.7.5.1 Jun 1 2004 09:43:13
DyeNorm_SelectMethod: Rank Consistency Filter
DyeNorm_IsBGPopnOLOn: OFF
DyeNorm_CorrMethod: Linear & Lowess
Ratio_Version: Version A.7.5.1 Jun 1 2004 09:43:43
Ratio_ErrorModel: Hybrid Model
Ratio_UseSurrogates: Use of surrogates turned on
Ratio_AutoEstimateAddErrorRed: Auto-estimation turned off
Ratio_AutoEstimateAddErrorGreen: Auto-estimation turned off
FeatureExtractor_Version: Version A.7.5.1 Jun 1 2004 09:45:57
|
| |
|
| Submission date |
Oct 09, 2009 |
| Last update date |
Oct 09, 2009 |
| Contact name |
Richard S Finn |
| E-mail(s) |
[email protected]
|
| Phone |
310-206-3180
|
| Fax |
310-586-6830
|
| Organization name |
UCLA
|
| Department |
Medicine-Hem/Onc
|
| Street address |
10833 Le Conte, 11-934 Factor Building
|
| City |
LA |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL7264 |
| Series (1) |
| GSE18496 |
Breast Cell Lines: Experimental vs. Mixed Reference |
|
