|
| Status |
Public on Dec 11, 2011 |
| Title |
HC2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lymphocytes Healthy Control Subject
|
| Organism |
Homo sapiens |
| Characteristics |
de-identified research code: 02020702
age: 46
gender: Female
|
| Treatment protocol |
Presence of disease (amyotrophic lateral sclerosis) compared to absence of disease (healthy control subjects with no presence of cancer, autoimmune disease or neurological disease)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation, quality control evaluation and microarray experiments were performed at Cogenics Inc. (Morrisville, NC). Total RNA was extracted from the cell samples at Cogenics Inc. (Morrisville, NC) using standard procedures: TRIzol extraction following manufacturer's instructions (Invitrogen Life Technologies Carlsbad, CA) and RNA purification using Rneasy Mini-kit (Qiagen, Valencia, CA).
quality control: The quantity of each of the total RNA samples (A260/280 ratio and yield) was determined by spectrophotometry and the size distribution (RIN) was assessed using an Agilent Bioanalyzer 2100.
|
| Label |
Cy5
|
| Label protocol |
Fifty nanograms of total RNA was converted into labeled cRNA with nucleotides coupled to a fluorescent dye (either Cy3 or Cy5) using the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
HC Reference pool HC1 to HC11
|
| Organism |
Homo sapiens |
| Characteristics |
reference: HC Reference pool
|
| Treatment protocol |
Presence of disease (amyotrophic lateral sclerosis) compared to absence of disease (healthy control subjects with no presence of cancer, autoimmune disease or neurological disease)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation, quality control evaluation and microarray experiments were performed at Cogenics Inc. (Morrisville, NC). Total RNA was extracted from the cell samples at Cogenics Inc. (Morrisville, NC) using standard procedures: TRIzol extraction following manufacturer's instructions (Invitrogen Life Technologies Carlsbad, CA) and RNA purification using Rneasy Mini-kit (Qiagen, Valencia, CA).
quality control: The quantity of each of the total RNA samples (A260/280 ratio and yield) was determined by spectrophotometry and the size distribution (RIN) was assessed using an Agilent Bioanalyzer 2100.
|
| Label |
Cy3
|
| Label protocol |
Fifty nanograms of total RNA was converted into labeled cRNA with nucleotides coupled to a fluorescent dye (either Cy3 or Cy5) using the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy3 and Cy5-labeled cRNA (825 ng) from two different samples (test and reference pool) were hybridized to Agilent Human Whole Genome 4x44k Microarrays.
|
| Scan protocol |
The hybridized arrays were washed and scanned using Agilent G2565BA scanner and data were extracted from the scanned image using Feature Extraction version 10.2 (Agilent Technologies).
|
| Description |
Pooled RNA from HC1 to HC11 for cohybridization with Cy5 labelled RNA
patient samples: Lymphocytes were isolated from whole blood drawn by venipuncture into BD ACD tubes using the method described by Repnik et al. (2003) [PMID:12957415].
|
| Data processing |
Conversion of raw data .txt files to .mev files, data normalization and filtering using the TM4 (MIDAS pipeline) were performed by the PI Jean-Luc Mougeot. Raw data .txt files in Agilent format were converted to .mev files using ExpressConverterTM v2.1 of the TM4 Microarray Suite (TIGR Genomics, Rockville, CA). Background-subtracted raw data were normalized using the MIDAS pipeline (TM4, TIGR Genomics, Rockville, MD) according to Sioson et al. (2006) [PMID: 16626497] with the following steps: total intensity normalization, LocFit (LOWESS), standard deviation regularization and low intensity trim. We generated two datasets for further analysis, DS3500 and DS7000 where both Cy3 and Cy5 integrated intensities (ISI) were above one (ISI=3500) or two (ISI=7000) standard deviation(s) of their respective background.
|
| |
|
| Submission date |
Mar 29, 2011 |
| Last update date |
Dec 11, 2011 |
| Contact name |
Jean-Luc C Mougeot |
| E-mail(s) |
jean-luc.mougeot@carolinas.org
|
| Organization name |
Carolinas Medical Center
|
| Department |
Internal Medicine
|
| Street address |
1542 Garden Terrace
|
| City |
Charlotte |
| State/province |
NC |
| ZIP/Postal code |
28232-2861 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE28253 |
Peripheral blood lymphocytes: ALS patients vs. healthy controls |
|
