|
| Status |
Public on Sep 28, 2007 |
| Title |
Vector_transduced_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from vector control transduced JSC1 PEL cells repeat 3 labelled with Cy5
|
| Organism |
Homo sapiens |
| Characteristics |
PEL cell line JSC1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Total RNA (25µg) was primed using anchored oligo(dT) and a pool of KSHV gene specific 3' primers (0.2 µmol), and reverse transcribed into Cy5-dCTP labelled cDNA using the CyScribe First Strand cDNA labelling kit (GE Healthcare) according to the manufacturer's protocol. Following cDNA labelling, residual RNA was denatured, and unincorporated nucleotides were removed by filtering through Microcon YM-30 columns (Millipore). Cy5-labelled cDNA was mixed 1:1 with Cy3-labelled cDNA synthesized from a custom-made reference RNA (batched mixture of TPA-induced JSC-1 and HeLa total RNA)
|
| |
|
| Channel 2 |
| Source name |
Common reference RNA labelled with Cy3
|
| Organism |
Homo sapiens |
| Characteristics |
to be added
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Total RNA (25µg) was primed using anchored oligo(dT) and a pool of KSHV gene specific 3' primers (0.2 µmol), and reverse transcribed into Cy5-dCTP labelled cDNA using the CyScribe First Strand cDNA labelling kit (GE Healthcare) according to the manufacturer's protocol. Following cDNA labelling, residual RNA was denatured, and unincorporated nucleotides were removed by filtering through Microcon YM-30 columns (Millipore). Cy5-labelled cDNA was mixed 1:1 with Cy3-labelled cDNA synthesized from a custom-made reference RNA (batched mixture of TPA-induced JSC-1 and HeLa total RNA)
|
| |
|
| |
| Hybridization protocol |
The array slides were post-processed and hybridized with the Cy5/Cy3 mix under coverslips in array hybridization chamber (Ambion) at 65oC for 20h
|
| Scan protocol |
Scanned using an Axon GenePix 4000B and GenePix and GenePix Pro 4.0.1.23 software
|
| Description |
Biological replicate 1 of 3
|
| Data processing |
Briefly, data were extracted from microarray image files using GENEPIX PRO 4.0 software (Axon Instruments). The signal to noise ratio (SNR) for individual spots was calculated by dividing the spot signal by the local background. The log2 median of ratios (Cy5/Cy3) were filtered to remove all flagged data and spots with SNR<2 in both the Cy3 and Cy5 channel. The median expression ratio from the quadruplicate spots of each array probe was used for subsequent analysis. The expression ratios generated from each array were assembled and filtered for genes present in all the arrays. The filtered data were median centered for both genes and arrays in Cluster (12) to normalize both the host and viral genes.
|
| |
|
| Submission date |
Sep 19, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Paul Kellam |
| E-mail(s) |
p.kellam@ucl.ac.uk
|
| Phone |
00442076799559
|
| URL |
http://www.ucl.ac.uk/medicalschool/infection-immunity/research/group-leaders/pkellam.htm
|
| Organization name |
UCL
|
| Department |
Infection
|
| Lab |
Virus Genomics & Bioinformatics
|
| Street address |
46 Cleveland Street
|
| City |
London |
| State/province |
London |
| ZIP/Postal code |
W1T 4JF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL5849 |
| Series (1) |
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