The Vero lineage was isolated from kidney epithelial cells extracted from African green monkey (Cercopithecus aethiops). The lineage was developed on 27 Mar 1962, by Yasumura and Kawakita at the Chiba University in Chiba, Japan.
Biomaterial provider
ATCC
Treatment protocol
Sample harvested at 10 day after BKV infection
Growth protocol
Vero cells, a line of African green monkey normal kidney epithelial cell (American Type Culture Collection, Manassas, Va.), were used throughout this study. Monolayer cultures of the cells were propagated in Eagle’s minimal essential medium (EMEM) (Mediatech Inc., Herndon, Va.), supplemented with 5% heat-inactivated fetal bovine serum (Mediatech Inc.) equilibrated with 5% CO2, in a humidified 37°C incubator. BK virus Gardner strain was kindly supplied by Dr. Walter Atwood (Brown University, Providence, RI) for use in infection experiments. Virus was processed and maintained as described previously(6). During infection procedure, Vero cells were grown at 37°C in T75 spinner flasks to a cell density of approximately 70% confluence prior to infection. Stocks of virus particles were appropriately diluted in EMEM culture medium supplemented with2% FBS/1% P/S in a volume sufficient to cover the cells. Infections were performed at a multiplicity of infection (MOI) of 5 PFU/cell at room temperature and virus was allowed to adhere to cells for one hour at 37°C, 5% CO2 with gentle rocking every 15 minutes to ensure that the inoculums were evenly distributed. After adsorption, complete media were then added and the infected cells were incubated at 37°C, 5% CO2 in a humidified incubator. At the indicated times post-infection (5, 10, 15, 19, and 25 dpi), the virus-containing culture medium was removed and the cells were washed twice gently with in phosphate buffered saline (PBS) directly in the flask. Remained PBS was taken away as far as possible and 2 ml of Trizol reagent (Invitrogen, Carlsbad, CA) were added into each flask. The infected cells were lysed entirely and harvested by pipetting up and down to lift the cells from the surface of flask. Finally, the cell lysates were transferred into 1.5-ml fresh microcentrifuge tubes on ice, and then frozen at -80°C until preparation of RNA. Mock-infected control cells were treated and harvested in the same manner as those infected, except that EMEM without virus was used during the infection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracte by lying directly in the 100mm petri dish with 2.0 ml TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction and then purified by RNeasy kit (Qiagen, Inc., Valencia, CA).
Label
Cy5
Label protocol
3DNA Array 50 kit was used for fluorescence labeling on cDNA. Each total RNA (20μg) sample was reversely transcribed by Superscript II to single strand cDNA with the capture primer. cDNA was then purified by microcon YM-30 column and was blocked by addictive 0.5μg of Cot1 human DNA.
The Vero lineage was isolated from kidney epithelial cells extracted from African green monkey (Cercopithecus aethiops). The lineage was developed on 27 Mar 1962, by Yasumura and Kawakita at the Chiba University in Chiba, Japan.
Biomaterial provider
ATCC
Treatment protocol
NO
Growth protocol
Vero cells, a line of African green monkey normal kidney epithelial cell (American Type Culture Collection, Manassas, Va.), were used throughout this study. Monolayer cultures of the cells were propagated in Eagle’s minimal essential medium (EMEM) (Mediatech Inc., Herndon, Va.), supplemented with 5% heat-inactivated fetal bovine serum (Mediatech Inc.) equilibrated with 5% CO2, in a humidified 37°C incubator. BK virus Gardner strain was kindly supplied by Dr. Walter Atwood (Brown University, Providence, RI) for use in infection experiments. Virus was processed and maintained as described previously(6). During infection procedure, Vero cells were grown at 37°C in T75 spinner flasks to a cell density of approximately 70% confluence prior to infection. Stocks of virus particles were appropriately diluted in EMEM culture medium supplemented with2% FBS/1% P/S in a volume sufficient to cover the cells. Infections were performed at a multiplicity of infection (MOI) of 5 PFU/cell at room temperature and virus was allowed to adhere to cells for one hour at 37°C, 5% CO2 with gentle rocking every 15 minutes to ensure that the inoculums were evenly distributed. After adsorption, complete media were then added and the infected cells were incubated at 37°C, 5% CO2 in a humidified incubator. At the indicated times post-infection (5, 10, 15, 19, and 25 dpi), the virus-containing culture medium was removed and the cells were washed twice gently with in phosphate buffered saline (PBS) directly in the flask. Remained PBS was taken away as far as possible and 2 ml of Trizol reagent (Invitrogen, Carlsbad, CA) were added into each flask. The infected cells were lysed entirely and harvested by pipetting up and down to lift the cells from the surface of flask. Finally, the cell lysates were transferred into 1.5-ml fresh microcentrifuge tubes on ice, and then frozen at -80°C until preparation of RNA. Mock-infected control cells were treated and harvested in the same manner as those infected, except that EMEM without virus was used during the infection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracte by lying directly in the 100mm petri dish with 2.0 ml TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction and then purified by RNeasy kit (Qiagen, Inc., Valencia, CA).
Label
Cy3
Label protocol
3DNA Array 50 kit was used for fluorescence labeling on cDNA. Each total RNA (20μg) sample was reversely transcribed by Superscript II to single strand cDNA with the capture primer. cDNA was then purified by microcon YM-30 column and was blocked by addictive 0.5μg of Cot1 human DNA.
Hybridization protocol
Two-step hybridizations were performed. During the first hybridization, cDNA products was pipetted onto the array. Then, the array was incubated in the hybridization chamber in a water bath at 65℃ for 16 to 20 hours for the probe/target hybridization. At the second hybridization, the 3DNA dendrimer was pipetted onto the array and the array was submerged in a 65℃ water bath for only 2 to 3 hours for capture primer – 3DNA dendrimer hybridization. After hybridization, array was washed with SSC and SDS mixed buffer suggested by manufacturer’s instruction of 3DNA array 50 kit.
Scan protocol
Microarrays were scanned by using GenePix 4100A (Axon, Foster City, CA) and images were taken at a resolution of 5μm. GenePix Pro 5.1 software was used to analyze image data.
Description
No
Data processing
Spots were first selected by (1) excluding spots defined as flag or absent (2) keeping only channels with signal - background / SD of background greater than 2 (3) keeping spot diameter greater 75μm (4) eliminating spots with intensity CV greater than 100 in ether channel. The log ratio of the last spots were calculated and normalized by pin-wised LOWESS fit normalization.
