|
Status |
Public on Dec 31, 2009 |
Title |
Sarcosine (duplicate) |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
untreated PrEC cells
|
Organism |
Homo sapiens |
Characteristics |
PrEC cells from Lonza (Cat No CC-0310)
|
Treatment protocol |
The cells are grown to 60-70% confluence and treated with 50 uM concentration of either alanine, leucine, glycine or sarcosine (Sigma, St Louis, MO), all resuspended in water for a period of 24h.
|
Growth protocol |
The cells are grown in Prostate Epithelial Cell Medium (Lonza Walkersville Inc. Walkersville, MD, Cat No CC-0310) supplemented with Bovine Pitutary extract, Hydrocortisone, human Epidermal Growth factor, Transferrin, Insulin, retinoic Acid, Triiodothyronine, and GA-1000 as per manufacturers instruction.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions.
|
Label |
cy3
|
Label protocol |
One ug of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
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|
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Channel 2 |
Source name |
Sarcosine treated PrEC cells, rep2
|
Organism |
Homo sapiens |
Characteristics |
50 uM sarcosine added to PrEC cells
|
Treatment protocol |
The cells are grown to 60-70% confluence and treated with 50 uM concentration of either alanine, leucine, glycine or sarcosine (Sigma, St Louis, MO), all resuspended in water for a period of 24h.
|
Growth protocol |
The cells are grown in Prostate Epithelial Cell Medium (Lonza Walkersville Inc. Walkersville, MD, Cat No CC-0310) supplemented with Bovine Pitutary extract, Hydrocortisone, human Epidermal Growth factor, Transferrin, Insulin, retinoic Acid, Triiodothyronine, and GA-1000 as per manufacturers instruction.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions.
|
Label |
cy5
|
Label protocol |
One ug of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
|
|
|
Hybridization protocol |
825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65 C hybridization oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to the manufacturer's recommnded protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent). Arrays were washed according to the manufacturer's recommended protocol including the Stabilization and Drying Solution step (Two-Color Microarray-Based Gene Expression Analysis, Agilent).
|
Scan protocol |
Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505B, Agilent) using the default settings for 4x44k format two-color arrays.
|
Description |
Samples 1 and 2 are technical replicates
|
Data processing |
Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software (Version 9.5.1.1). Spot values were normalized using the default linear-lowess normalization. Samples M5a and M5b were isolated from the same patient and normalized values were averaged before data analysis.
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|
|
Submission date |
Jan 14, 2008 |
Last update date |
Dec 31, 2008 |
Contact name |
Laila M Poisson |
E-mail(s) |
lpoisso1@hfhs.org
|
Organization name |
Henry Ford Health Systems
|
Department |
Biostatistics and Research Epidemiology
|
Street address |
1 Ford Place
|
City |
Detroit |
State/province |
MI |
ZIP/Postal code |
48202 |
Country |
USA |
|
|
Platform ID |
GPL4133 |
Series (1) |
GSE10164 |
PrEC response to Alanine or Sarcosine at 6 hours |
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