|
| Status |
Public on Dec 30, 2011 |
| Title |
BC-1 lytic tech rep |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BC-1: KSHV-positive, EBV-positive Primary Effusion Lymphoma
|
| Organisms |
Homo sapiens; human gammaherpesvirus 4; Human gammaherpesvirus 8 |
| Characteristics |
cell type: KSHV-positive, EBV-positive Primary Effusion Lymphoma
cell line: BC-1
treatment: Lytic-induction = tissue culture medium supplemented with 20ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA) + 0.25mM sodium butyrate (NaB) for 48hrs
replicate: 2
array: 8
|
| Treatment protocol |
No additional treatments
|
| Growth protocol |
Cells were grown in Roswell Park Memorial Institute formulation 1640 media with 10% fetal bovine serum. Growth medium is supplemented with 200 U/mL penicillin and 200 μg/mL streptomycin. Cells were grown at 37 °C with a 5% CO2 humidified atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) as described [Chomczynski P, Sacchi N: The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162(1):156-159]. RNA was precipitated using the nucleating agent linear acrylamide (Ambion, Austin, TX) as described [Gaillard C, Strauss F: Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res 1990, 18(2):378]. RNA samples were treated with Turbo DNase (Ambion, Austin, TX) to remove contaminating genomic DNA as per manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
150 ng of RNA was reverse transcribed with an oligo d(T) promoter primer into cDNA using the Agilent Quick Amp Kit, Two Color per manufacturer's protocol. The cDNA was then transcribed into cRNA containing Cy3 or Cy5 using Agilent T7 RNA polymerase.
|
| |
|
| Channel 2 |
| Source name |
BJAB: EBV-negative, KSHV-negative Burkitt Lymphoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: EBV-negative, KSHV-negative Burkitt Lymphoma
cell line: BJAB
treatment: Untreated = standard tissue culture conditions
replicate: 2
array: 8
|
| Treatment protocol |
No additional treatments
|
| Growth protocol |
Cells were grown in Roswell Park Memorial Institute formulation 1640 media with 10% fetal bovine serum. Growth medium is supplemented with 200 U/mL penicillin and 200 μg/mL streptomycin. Cells were grown at 37 °C with a 5% CO2 humidified atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) as described [Chomczynski P, Sacchi N: The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162(1):156-159]. RNA was precipitated using the nucleating agent linear acrylamide (Ambion, Austin, TX) as described [Gaillard C, Strauss F: Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res 1990, 18(2):378]. RNA samples were treated with Turbo DNase (Ambion, Austin, TX) to remove contaminating genomic DNA as per manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
150 ng of RNA was reverse transcribed with an oligo d(T) promoter primer into cDNA using the Agilent Quick Amp Kit, Two Color per manufacturer's protocol. The cDNA was then transcribed into cRNA containing Cy3 or Cy5 using Agilent T7 RNA polymerase.
|
| |
|
| |
| Hybridization protocol |
Equal masses of Cy3- and Cy5-labeled samples were co-hybridized to microarrays.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner. Images were quantified using Agilent Feature Extraction Software (version 9.1.3.1).
|
| Description |
BC-1: KSHV-positive, EBV-positive, lytic, technical replicate
|
| Data processing |
Agilent Feature Extraction software (version 9.1.3.1) was used to generate normalized, background subtracted signal values ([g/r]ProcessedSignal). These values were used for log ratios of Cy3 to Cy5: Log10 (gProcessedSignal / rProcessedSignal). Saturated flag information was maintained for downstream analysis. LIMMA in-array analysis was performed using triplicate features per unique probe sequence after normalizing to Cy3 and Cy5 signal values to mean signal values of cellular housekeeping genes (ACTA1, GAPDH, and TUBA1B). The P-values for differential expression between the two channels are computed with Limma using the three triplicate probes per feature, and then adjusted for multiple testing according to Benjamini and Hochberg’s false discovery rate method (Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. JR Statist Soc B 1995, 57(1):289-300). Only adjusted P-values are indicated with the raw data, while the remaining calculations are available in the supplement of the associated article. Cross-array LIMMA analysis is also reported in the supplement of the associated article.
|
| |
|
| Submission date |
Dec 19, 2011 |
| Last update date |
Dec 30, 2011 |
| Contact name |
Lindsay Renee Dresang |
| E-mail(s) |
lid26@pitt.edu
|
| Organization name |
University of Pittsburgh
|
| Department |
Cancer Virology Program
|
| Lab |
Chang Moore
|
| Street address |
5117 Centre Ave, Hillman Cancer Res. Pav. 1.8
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL15030 |
| Series (1) |
| GSE34538 |
Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes |
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