|
| Status |
Public on Apr 09, 2013 |
| Title |
AG14778_sham_C5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Non AT carrier
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: AG14778
cell type: lymphoblastoid
genotype: ATM+/+
|
| Biomaterial provider |
Coriell; http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=AG14778
|
| Growth protocol |
Cells were cultured in RPMI supplemented with 15% FBS (Life Technologies). All cell lines obtained from the NIGMS were maintained at 37oC in a humidified atmosphere of 5% CO2 and were routinely tested and shown to be free of mycoplasma contamination using a commercial assay
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kit (Qiagen Sciences). The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy5
|
| Label protocol |
1 μg of sample RNA and global reference RNA were converted to cDNA with reverse transcriptase and then amplified using T7 RNA polymerase while labeling with either cyanine 3 deoxyuridine triphosphate (Cy3-dUTP) or Cy5-dUTP. The quality of each labeled cRNA was evaluated using an Agilent 2100 bioanalyzer before hybridization; 750 ng of Cy3- and Cy5-labeled cRNA were used in the hybridization.
|
| |
|
| Channel 2 |
| Source name |
Global reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
source: Stratagene
|
| Growth protocol |
Cells were cultured in RPMI supplemented with 15% FBS (Life Technologies). All cell lines obtained from the NIGMS were maintained at 37oC in a humidified atmosphere of 5% CO2 and were routinely tested and shown to be free of mycoplasma contamination using a commercial assay
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kit (Qiagen Sciences). The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
1 μg of sample RNA and global reference RNA were converted to cDNA with reverse transcriptase and then amplified using T7 RNA polymerase while labeling with either cyanine 3 deoxyuridine triphosphate (Cy3-dUTP) or Cy5-dUTP. The quality of each labeled cRNA was evaluated using an Agilent 2100 bioanalyzer before hybridization; 750 ng of Cy3- and Cy5-labeled cRNA were used in the hybridization.
|
| |
|
| |
| Hybridization protocol |
The labeled cRNA from human lymphoblastoid cells was hybridized with the labeled global reference cRNA on an Agilent array in a hybridization oven at 60°C for 17 hr.
|
| Scan protocol |
Scanned on an Agilent Technologies Scanner G2505B US23502387
Images were quantified using Agilent Feature Extraction Software (version A.7.1.1).
|
| Description |
cell lines with same ATM genotype receiving the same treatment (Sham or IR) are biological replicates of one-another
|
| Data processing |
Each RNA sample was subjected to microarray analysis twice with C3/C5 dyes swap (C3 labeled test sample / C5 labeled reference, C5 labeled test sample /C3 labeled reference) in both Basal and IR study. The data from each array were normalized using EPIG method, which included array-based systematic variation normalization [Chou, J.W., Paules, R.S., and Bushel, P.R. (2005). Systematic variation normalization in microarray data to get gene expression comparison unbiased. J. Bioinform. Comput.Biol. 3, 225-241], profile-based C3/C5 dye-swap correction, and biological reference state alignment [Chou, J.W., Zhou, T., Kaufmann, W. K., Paules, R.S., and Bushel, P.R. (2007). Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes. BMC Bioinformatics 8, 427. doi:10.1186/1471-2105-8-427].
|
| |
|
| Submission date |
Apr 08, 2013 |
| Last update date |
Apr 26, 2013 |
| Contact name |
Tong Zhou |
| E-mail(s) |
tong.zhou@gentris.com
|
| Phone |
919-724-9814
|
| Organization name |
Gentris Corperation
|
| Department |
Clincial Genetics
|
| Street address |
133 Southcenter Court, Suite 400
|
| City |
Morrisville |
| State/province |
NC |
| ZIP/Postal code |
27560 |
| Country |
USA |
| |
|
| Platform ID |
GPL7260 |
| Series (2) |
| GSE45849 |
Gene expressioin signatures in human lymphoblastoid cell lines with different ataxia telangiectasia-mutated (ATM) genotypes post sham- or IR-treatment |
| GSE45850 |
Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals and AT patients |
|
