|
| Status |
Public on Oct 29, 2011 |
| Title |
LCL MZ 49a |
| Sample type |
RNA |
| |
|
| Source name |
Whole blood was collected directly into ACD (Acid Citrate Dextrose) vacuum tube. Mononucleated cells were isolated using a ficoll gradient and LCL established by Epstein-Barr virus transformation of lymphocytes and stored in liquid nitrogen.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Lymphoblastoid Cell Line
twin: 49a
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole blood was collected directly into ACD (Acid Citrate Dextrose) vacuum tube. Mononucleated cells were isolated using a ficoll gradient and LCL established by Epstein-Barr virus transformation of lymphocyte and stored in liquid nitrogen. For RNA isolation, established cell lines were regrown under tightly controlled growth conditions in the same batch of RPMI 1640 media with 10% FCS and antibiotics, to limit the cell culture effects on RNA preparation. Total RNA was extracted from samples using Qiagen RNeasy Midi-Kits (QIAGEN, Valencia, CA), when cells were in log phase growth.
|
| Label |
NA
|
| Label protocol |
standard Illumina protocol
|
| |
|
| Hybridization protocol |
Expression profiles were generated by hybridising 750ng of cRNA to Illumina HumanHT-12 v3.0 Beadchip according to Illumina whole-genome gene expression direct hybridization assay guide (Illumina Inc, San Diego, USA). Briefly, 500 ng of total RNA were used to generate biotinylated cRNA, which was fragmented and hybridised to an Illumina whole genome expression chip, HumanHT-12 v3.0 for 18 h at 58oC.
|
| Scan protocol |
Beadchips were then washed and stained and subsequently scanned to obtain fluorescence intensities using an Illumina Bead Array Reader (BAR). A Latin square design was used to randomise the samples on the chips and chip positions.
|
| Description |
Whole blood samples were collected from MZ twin pairs and processed within 24 hours of collection
|
| Data processing |
Relative expression values were generated for each transcript using Illumina Genome Studio software (Illumina Inc., San Diego). To minimise the influence of overall signal levels, which may reflect RNA quantity and quality rather than a true biological difference between individuals, the following standardisation procedures were used. Background noise detected from negative control beads was subtracted from raw expression values for each transcript. Data were then filtered for gene transcripts that were present in at least 50% of samples at p < 0.05 according to the global-error threshold calculated by Genome Studio’s cross-gene error model. To prevent the introduction of bias between the LCL and WB samples, the raw microarray data from both tissue samples were quantile normalised together. Adjusted expression levels for each transcript were transformed using a quantile transformation to achieve a stabilized variance distribution across average expression levels.
Non-normalised data have just background subtraction from the raw signals. Normalised data has been filtered for genes significantly expressed in greater than 50% of samples. Values have then been quantile normalized.
|
| |
|
| Submission date |
Oct 28, 2011 |
| Last update date |
Oct 29, 2011 |
| Contact name |
Joseph E Powell |
| E-mail(s) |
joseph.powell@uq.edu.au
|
| Organization name |
University of Queensland
|
| Department |
Queensland Brain Institute
|
| Street address |
St lucia
|
| City |
Brisbane |
| State/province |
Qld |
| ZIP/Postal code |
4006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL6947 |
| Series (1) |
| GSE33321 |
Genetic control of gene expression in whole blood and lymphoblastoid cell lines is largely independent |
|
