|
| Status |
Public on May 28, 2009 |
| Title |
Whole population (jhu-251485011211-4.txt_Cy3) |
| Sample type |
RNA |
| |
|
| Source name |
BULK_1; Cy3; whole population of sw780 tumor
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: 67 Positive and Negative
disease state: urothelial cancer
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using RNAzol (Invitrogen), followed by a secondary purification using the RNEasy system (Qiagen). RNA integrity was determined the Agilent 2100 Bioanalyzer. RNA concentration for individual samples was > 250 ng/µl, with 28S/18S ratio > 1.4 and RIN score > 9.4.
|
| Label |
Cy3
|
| Label protocol |
Sample amplification and labeling procedures for microarray analysis were carried out by using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). 400 ng of total RNA was reverse transcribed into first strand and second strand cDNA by MMLV-RT using an oligo dT primer (System Biosciences) that incorporates a T7 promoter sequence. The cDNA was then used as a template for in vitro transcription in the presence of T7 RNA polymerase and Cyanine labeled CTPs. The labeled cRNA was purified using RNeasy mini kit (Qiagen), followed by quantification based on both concentrations of cRNA and dye. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification and labeling according to manufacturer`s protocol.
|
| |
|
| Hybridization protocol |
1.5 µg of each sample labeled with Cy3 or Cy5 was mixed with control targets (Agilent Technologies). Fragmentation was carried out by incubating for 30 minutes at 60 degrees Celsius and stopped by adding an equal volume of 2x hybridization bu?er (Agilent Technologies). Fragmented targets were added to the microarrays, assembled into a hybridization chamber (Agilent Technologies) and hybridized at 65 degrees Celsius for 17 hours in a hybridization oven with rotation. Hybridized microarrays were washed and dried according to the Agilent microarray processing protocol.
|
| Scan protocol |
Hybridized slides were scanned using a G2565BA Agilent scanner, with 100% PMT and 10 micrometer resolution according to the manufacturer`s directions. The scanner was operated by the Agilent Scan Control 7.0 software. Data were extracted with the Agilent Feature Extraction 9.5.3.1 software. Images were subjected to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of red and green channels, both for foreground and background, to identify anomalous arrays. No abnormalities were found and all arrays were retained for subsequent analyses.
|
| Description |
MHC1-Negative
|
| Data processing |
Within-array dye e?ects were corrected by the ?loess? normalization method. The ?scale? normalization method was applied to standardize the M- and the A-values distributions across different arrays, by using the median-absolute-value as scale estimator. No background subtraction was performed prior to normalization. Positive and negative microarray control features were not used to compute the ?loess? smoothing and were further excluded from the subsequent analysis. After scaling the normalized A and M values were deconvoluted into single channel intensities
|
| |
|
| Submission date |
May 27, 2009 |
| Last update date |
Nov 15, 2012 |
| Contact name |
Luigi Marchionni |
| E-mail(s) |
marchion@jhu.edu
|
| Phone |
410-502-8179
|
| Organization name |
Johns Hopkins University
|
| Department |
Oncology
|
| Lab |
Cancer Biology Program
|
| Street address |
1550 Orleans St., CRB2, Room 1M52
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE16255 |
Gene expression in a Highly Tumorigenic Cells (HTC) purified from Urothelial Cancer |
|
