|
| Status |
Public on Aug 17, 2009 |
| Title |
control vs. glutamine alone_rep 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BxPC-3 pancreatic cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
growth condition: Glucose and glutamine depleted (control)
|
| Growth protocol |
BxPC-3 cells were plated in quadruplicate and starved overnight in glucose and glutamine free media. For glucose and/or glutamine induction, cells were incubated for 6hrs in media containing glucose (25mM) or glutamine (2mM) or glucose plus glutamine (25mM and 2mM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNAeasy minikit from qiagen
|
| Label |
Cy3
|
| Label protocol |
The Agilent Two-Color Low RNA Input Linear Amplification Kit is used to generate fluorescently labeled cRNA for two-color microarray hybridizations. Agilent RNA spike-in controls are combined with input total RNA samples (50 to 500 ng). The polyadenylated fraction of the total RNA sample is primed with oligo dT/T7 RNA polymerase promoter oligonucleotide sequences and cDNA synthesis is accomplished through the addition of MMLV-RT. Following cDNA synthesis, T7 RNA polymerase and dye-labeled nucleotides are combined with the reaction mixture to simultaneously amplify the target material through the generation of cRNA and incorporate either cyanine 3-CTP or cyanine 5-CTP. Fluorescently labeled, cRNA molecules are purified from the reaction mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is determined using a NanoDrop ND-1000 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
BxPC-3 pancreatic cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
growth condition: Glutamine alone
|
| Growth protocol |
BxPC-3 cells were plated in quadruplicate and starved overnight in glucose and glutamine free media. For glucose and/or glutamine induction, cells were incubated for 6hrs in media containing glucose (25mM) or glutamine (2mM) or glucose plus glutamine (25mM and 2mM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNAeasy minikit from qiagen
|
| Label |
Cy5
|
| Label protocol |
The Agilent Two-Color Low RNA Input Linear Amplification Kit is used to generate fluorescently labeled cRNA for two-color microarray hybridizations. Agilent RNA spike-in controls are combined with input total RNA samples (50 to 500 ng). The polyadenylated fraction of the total RNA sample is primed with oligo dT/T7 RNA polymerase promoter oligonucleotide sequences and cDNA synthesis is accomplished through the addition of MMLV-RT. Following cDNA synthesis, T7 RNA polymerase and dye-labeled nucleotides are combined with the reaction mixture to simultaneously amplify the target material through the generation of cRNA and incorporate either cyanine 3-CTP or cyanine 5-CTP. Fluorescently labeled, cRNA molecules are purified from the reaction mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is determined using a NanoDrop ND-1000 spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Fluorescently labeled cRNA samples (825 ng each) were fragmented and combined with Agilent Hi-RPM Hybridization Buffer. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization oven and were incubated at 65ºC for 17 hours with a rotational speed of 10 rpm. Following incubation, the microarray slide was washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; room temperature) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; 31ºC) for 1 minute each. Microarray slides were briefly dipped in a solution of acetonitrile and dried.
|
| Scan protocol |
Microarray slides were scanned in an Agilent Technologies G2505B Microarray Scanner at 5 µm resolution using the Extended Dynamic Range scanning protocol.
Images were processed using Agilent Feature Extraction software version 9.5.1.1.
|
| Description |
no additional information
|
| Data processing |
LOWESS normalized log ratios reported by the Agilent platform were converted to log base 2.
|
| |
|
| Submission date |
Aug 13, 2009 |
| Last update date |
Aug 13, 2009 |
| Contact name |
Mohan Rao Kaadige |
| E-mail(s) |
mohan.kaadige@hci.utah.edu
|
| Phone |
801-585-5169
|
| Fax |
801-585-6410
|
| Organization name |
Huntsman Cancer Institute
|
| Department |
Oncological Sciences
|
| Lab |
Don Ayer
|
| Street address |
2000 Circle of Hope
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE17632 |
BxPC-3 cells: growth in different nutrients |
|
