|
| Status |
Public on Jan 04, 2008 |
| Title |
EBV positive Burkitt's lymphoma cell line Akata, control vs miR-146a infection pair 1 - Dye Swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Akata LMP1 (pEhyg- miR-146a) infection 1
|
| Organism |
Homo sapiens |
| Characteristics |
Akata cells were then infected with a retrovirus encoding miR-146a (pEhyg-miR-146a). This sample represents infection 1 of two separate infections with pEhyg-miR-146a.
|
| Biomaterial provider |
Erik Flemington
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen total RNA purification kit - Standard conditions
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
Akata control (pEhyg) infection 1
|
| Organism |
Homo sapiens |
| Characteristics |
Akata cells were then infected with a control retrovirus (pEhyg-). This sample represents infection 1 of two separate infections with pEhyg.
|
| Biomaterial provider |
Erik Flemington
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen total RNA purification kit - standard conditions
|
| Label |
Cy5
|
| Label protocol |
For the linear T7-based amplification step, 1 μg of each total RNA sample was used as starting material. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole Human Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven.
Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% Nlauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner (Agilent Technologies).
|
| Description |
Dual color scanning
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (p-values). For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware).
VALUE = LogRatio (base 10) - log(miR-146a/control) per feature (processed signals used)
|
| |
|
| Submission date |
Jan 02, 2008 |
| Last update date |
Jan 03, 2008 |
| Contact name |
Erik K Flemington |
| E-mail(s) |
eflemin@tulane.edu
|
| Phone |
504 988-1167
|
| Organization name |
Tulane Health Sciences Center
|
| Department |
Pathology
|
| Street address |
1430 Tulane Ave., SL79
|
| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70112 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (2) |
| GSE10057 |
The Epstein-Barr Virus latent membrane protein 1 (LMP1) induces cellular microRNA146a |
| GSE10107 |
The EBV latent membrane protein 1 (LMP1) induces cellular microRNA-146a, a modulator of lymphocyte signaling pathways |
|
