|
Status |
Public on Mar 08, 2007 |
Title |
MA-10 stably transfected with human LHR mutation Asp578Gly 3 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
MA-10 Asp578Gly 3
|
Organism |
Mus musculus |
Characteristics |
MA-10 stably transfected with human LHR mutation Asp578Gly
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted by Trizol reagent (invitrogen).
|
Label |
Cy3
|
Label protocol |
An indirect labeling method using aminoallyl-dUTP and amino-reactive fluorescent dyes overnight.
|
|
|
Channel 2 |
Source name |
MA-10 cell
|
Organism |
Mus musculus |
Characteristics |
MA-10 cell
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted by Trizol reagent (invitrogen).
|
Label |
Cy5
|
Label protocol |
An indirect labeling method using aminoallyl-dUTP and amino-reactive fluorescent dyes overnight.
|
|
|
|
Hybridization protocol |
Microarrays were prehybridized in 1% bovine serum albumin, 5× SSC, 0.1% SDS for 45 min at 42°C, washed in H2O and dried by centrifugation. Cy3 and Cy5 labeled cDNA samples were mixed with 50 μl of hybridization buffer and applied to the microarrays for hybridization at 37°C for 16 hours in a hybridization chamber in the dark with gentle agitation. Slides were washed for 10 minutes at 50°C in 1× SSC and 0.1% SDS in shaking incubator, followed by a 1 minute wash in 1× SSC, three 1 minute washes in 0.1× SSC, and one rinse in H2O, at room temperature. Slides were dried by centrifugation.
|
Scan protocol |
Images were acquired for Cy3 and Cy5 channels in a 16-bit TIFF format using ScanArray Express v2.2 software. Laser power and the gain on the photomultiplier tube (PMT) were kept constant during scanning of each individual slide. The PMT setting was chosen so that the highest intensity values lied below the saturation point. Spot intensity and background signals were quantified for each channel and used to generate spreadsheet data for Microsoft Excel.
|
Description |
cDNA from the subject (Cy3) was co-hybridized with control MA-10 cDNA labelled with (Cy5). The ratio between Cy3 and Cy5 signal was normalized and in log scale.
|
Data processing |
The acquired image was processed in ScanArray Express v2.2 software. All bad spots were flagged. Data obtained from each hybridization were stored in a database for analysis. Cy3/Cy5 ratios were normalized with respect to the median ratio value of all of the spots in the array. After normalization, spots with intensities for both channels (sum of medians) less than that of the local background were discarded. The ratios of the remaining spots were log transformed (base 2), and duplicated spots on the OncoChip were averaged to the median. Biological functions of selected genes were assigned by Gene Ontology databases.
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|
|
Submission date |
Jan 18, 2007 |
Last update date |
Feb 08, 2007 |
Contact name |
TIN-LAP LEE |
Organization name |
National Institutes of Health
|
Department |
NICHD
|
Lab |
Laboratory of Clincal Genomics
|
Street address |
2C08 Building 49
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL4755 |
Series (1) |
GSE6780 |
Evaluating the effects of activating mutations of human LH receptor in MA-10 cell |
|