|
| Status |
Public on Apr 03, 2008 |
| Title |
12hour-A ds |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
vsf- HSV infected 12hour-A dye-swap
|
| Organism |
Mus musculus |
| Characteristics |
MEF from 129 Sv/Ev mice at embryonic day 24
vsf-null HSV infected MEF cells
|
| Treatment protocol |
Cultures of 2.5x106 5 MEF cells in 100mm dishes were infected at an MOI of 5 with KOS or Dvhs1 for 30 min followed by removal of inoculum and addition of complete medium.
|
| Growth protocol |
Viral stocks were grown and titered on Vero cells. Mouse embryo fibroblast (MEF) cultures were generated from 129 Sv/Ev mice at embryonic day 15, and passaged once before being plated for infection. MEFs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.1 mM sodium pyruvate, 250 U/mL penicillin, 250 μg/mL streptomycin, and 250 ng/mL amphotericin B.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified and DNase-treated with the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA quality was determined by Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer’s recommendations.
|
| Label |
Cy5
|
| Label protocol |
First strand cDNA was generated by random and oligo dT primed reverse transcription (Superscript II, Invitrogen, Carlsbad, CA) utilizing the 3DNA Array 900MPX kit (Genisphere, Hatfield, PA), according to the manufacturer’s protocol. The cDNAs were quantitated via spectrophotometry to confirm recovery. Samples were paired and balanced by mass.
|
| |
|
| Channel 2 |
| Source name |
wt HSV infected 12hour-A dye-swap
|
| Organism |
Mus musculus |
| Characteristics |
MEF from 129 Sv/Ev mice at embryonic day 24
wild type HSV infected MEF cells
|
| Treatment protocol |
Cultures of 2.5x106 5 MEF cells in 100mm dishes were infected at an MOI of 5 with KOS or Dvhs1 for 30 min followed by removal of inoculum and addition of complete medium.
|
| Growth protocol |
Viral stocks were grown and titered on Vero cells. Mouse embryo fibroblast (MEF) cultures were generated from 129 Sv/Ev mice at embryonic day 15, and passaged once before being plated for infection. MEFs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.1 mM sodium pyruvate, 250 U/mL penicillin, 250 μg/mL streptomycin, and 250 ng/mL amphotericin B.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified and DNase-treated with the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA quality was determined by Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer’s recommendations.
|
| Label |
Cy3
|
| Label protocol |
First strand cDNA was generated by random and oligo dT primed reverse transcription (Superscript II, Invitrogen, Carlsbad, CA) utilizing the 3DNA Array 900MPX kit (Genisphere, Hatfield, PA), according to the manufacturer’s protocol. The cDNAs were quantitated via spectrophotometry to confirm recovery. Samples were paired and balanced by mass.
|
| |
|
| |
| Hybridization protocol |
Each sample pair was suspended in SDS-based hybridization buffer (Genisphere) and Array 50dT blocker (Genisphere). Two hybridizations were carried out in a sequential manner. The primary hybridization was performed under a supported glass coverslip (Erie Scientific, Portsmouth, NH) at 62ºC for 16-20 hrs. Prior to the secondary hybridization, slides were washed as described in the manufacturer’s protocol. Secondary hybridization was carried out using the 3DNA Array 350 kit (Genisphere) according to the manufacturer’s protocol, washed and t 1 reated with Dyesaver (Genisphere) before scanning.
|
| Scan protocol |
Slides were scanned immediately following hybridization on a ScanArray Express HT scanner (PerkinElmer, Waltham, MA). Laser power was kept constant, and photomultiplier tube values were set for optimal intensity with minimal background.
|
| Description |
Herpes simplex virus infected mouse embryo fibroblasts. Wild type (KOS) virus is co-hybridized with vhs null virus (NHB). Each time-point is hybridized in quadruplicate.
|
| Data processing |
Gridding and analysis of images were performed with Scanarray software express v3.0 (PerkinElmer). The resulting median pixel values were imported into Genespring v7.3 software (Agilent), and local background intensities were subtracted from individual spot intensities. To account for dye swap, the ‘signal’ channel and ‘control’ channel measurements were reversed in such samples so that signal derived from Dvhs-infected MEF RNA occupied the ‘signal’ channel and KOS-infected MEF RNA the ‘control’ channel. The mean signal and control intensities of the on-slide duplicate spots were calculated, and the control values were normalized with GeneSpring’s “Normalize to Positive Control Genes” function using 4 SpotReport® Oligo Array Validation System spike-in controls. If the control channel was lower than 10 relative fluorescence units (rfu), then 10 was used instead. Signal-to-normalized controlled ratios were calculated and the cross-chip averages were derived from the antilog of the mean of the natural log ratios across all biologic replicates. Oligonucleotide elements that received a detectable call (intensity >200 rfu or local signal-to-background >2 in at least one channel) by Scanarray software in either the Cy3 or Cy5 in at least 2 of 4 samples of at least 1 time-point were identified, and all others were excluded from the analysis. Genes were not filtered based on significance in order to be able to visualize global trends. Differences in dye incorporation and quantum yield were normalized by addition of A. thaliana mRNA spikes into the reverse 1 transcription reaction in equal molar amounts along with the experimental mRNA. Data represent two independent biological experiments with dye swaps such that two technical experiments were performed for each independent biological sample.
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| |
|
| Submission date |
Mar 27, 2008 |
| Last update date |
Mar 31, 2008 |
| Contact name |
Seth Daniel Crosby |
| E-mail(s) |
scrosby@wustl.edu
|
| Phone |
314-286-1256
|
| Organization name |
Washington Univeristy School of Medicine
|
| Department |
Genetics
|
| Lab |
GTAC
|
| Street address |
660 S. Euclid
|
| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL6599 |
| Series (1) |
| GSE10962 |
Herpes simplex host shutoff attenuates antiviral state |
|
