Vero-rKSHV.219 cells are Vero cells infected with recombinant Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus.
Biomaterial provider
Jeffrey Vieira, Ph.D. Seattle, WA, USA
Treatment protocol
For transfection, cells were seeded at 1x10^6 per 10-cm plate to obtain ~50 to 70% confluence the following day, and transfected with empty ER vector using TransIT-LT1 transfection reagent with 10 ug DNA according to the manufacturer’s (Mirus) instructions. Hygromycin B (300 ug/mL; Invitrogen) was added to cultures 3 hours post-transfection, and 4-hydroxytamoxifen (4-OHT; 0.1 mM; Sigma), was added 15 hours post-hygromycin. Total RNA was harvested 15 hours following 4-OHT addition.
Growth protocol
Vero-rKSHV.219 cells were grown in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 6ug/ml puromycin as described in Vieira and O'Hearn (Virology 325; 225-240).
Extracted molecule
total RNA
Extraction protocol
Extraction and purification by RNABee (Tel-test), as recommended by manufacturer.
Label
cyanine 5-dUTP [Cy5]
Label protocol
3 or 6 ugs. of each RNA sample (equal amounts per pair) were adjusted to 11 uL with dH2O. 2.2 ul of random hexamers (2 ug/ul; Sigma-Genosys) were added, the mixture was heated at 98°C for 2 minutes, then snapped cool on ice. 11.1 uL of master mix (5.0 uL First-strand buffer, 2.5 uL 100mM DTT, 2.3 uL low dTdNTP mix [5mM A,G,C and 0.2mM T stock], containing 1.5 uL cyanine 5-dUTP [Cy5] [PerkinElmer]) was added. The mixture was subsequently supplemented with 1.2 uL Superscript (Invitrogen), then incubated for 10 minutes at 25°C, followed by 90 min at 42°C.
Vero-rKSHV.219 cells are Vero cells infected with recombinant Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus.
Biomaterial provider
Jeffrey Vieira, Ph.D. Seattle, WA, USA
Treatment protocol
For transfection, cells were seeded at 1x10^6 per 10-cm plate to obtain ~50 to 70% confluence the following day, and transfected with empty ER vector using TransIT-LT1 transfection reagent with 10 ug DNA according to the manufacturer’s (Mirus) instructions. Hygromycin B (300 ug/mL; Invitrogen) was added to cultures 3 hours post-transfection. Total RNA was harvested 30 hours following hygromycin addition.
Growth protocol
Vero-rKSHV.219 cells were grown in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 6ug/ml puromycin as described in Vieira and O'Hearn (Virology 325; 225-240).
Extracted molecule
total RNA
Extraction protocol
Extraction and purification by RNABee (Tel-test), as recommended by manufacturer.
Label
cyanine 3-dUTP [Cy3]
Label protocol
3 or 6 ugs. of each RNA sample (equal amounts per pair) were adjusted to 11 uL with dH2O. 2.2 ul of random hexamers (2 ug/ul; Sigma-Genosys) were added, the mixture was heated at 98°C for 2 minutes, then snapped cool on ice. 11.1 uL of master mix (5.0 uL First-strand buffer, 2.5 uL 100mM DTT, 2.3 uL low dTdNTP mix [5mM A,G,C and 0.2mM T stock], containing 1.5 uL cyanine 3-dUTP [Cy3] [PerkinElmer]) was added. The mixture was subsequently supplemented with 1.2 uL Superscript (Invitrogen), then incubated for 10 minutes at 25°C, followed by 90 min at 42°C.
Hybridization protocol
Microcon 10 columns (YM 10; Millipore) were primed with 400 uL 1x TE buffer (Tris-EDTA pH 8.0) by centrifugation at RT for 10 min. Paired Cy3-labeled and Cy5-labeled cDNAs were combined, added to the primed column, and purified by centrifugation. When approximately 25 uL of liquid remained above the membrane, an extra 200 uL 1x TE buffer was added to wash the column. Centrifugation was continued until approximately 4.5 uL liquid remained above the membrane. The purified, labeled cDNAs were transferred to a new tube and combined with an equal volume of hybridization mixture (0.5 uL 10mg/ml tRNA (Sigma), 0.5 uL 10mg/ml salmon sperm DNA (Sigma), 1.0 uL 20x SSC, 2.5 uL formamide (Fisher), and 1.0 uL 1% SDS). The mixture was heated for 2 min at 98°C, and cooled for 5 min at RT immediately before loading on the microarray slides. Prior to hybridization, the slides were blocked in pre-hybridization solution (3.0 g bovine serum albumin [fraction V; Roche], 1.2ml 10% SDS, 106.0 ml Milli-Q water) at 42°C for 1 h. The pre-hybridized slides were washed with nuclease-free water for 2 min, isopropanol for 2 min, and subsequently dried by centrifugation. The labeled cDNAs were applied to slides, covered by a 22 x 22-mm cover-slip (Corning), sealed in a hybridization chamber (GeneMachines), and hybridized at 50°C overnight. After hybridization, cover-slips were removed by submerging the slides in solution I (2x SSC, 1% SDS), rinsed in solution II (1x SSC, 0.05%SDS) for 1 min, and rinsed in solution III (0.06x SSC) for 2 min.
Scan protocol
Slides were dried by centrifugation and scanned immediately with a GenePix4000B scanner (Molecular Devices). The images were processed using GenePix 5.1. Data were filtered by removing all spots that were below the background noise or flagged as ‘bad’. Spots were considered to be below the background noise if the sum of the median intensities of the two channels was less than twice the highest mean background of the chip. The chips were normalized by the print-tip Lowess method.
Description
none
Data processing
The ratio of the mean median intensity of Cy5 over the mean median intensity of Cy3 was determined for each spot.
Dye ratios for viral genes from the same transfections that were hybridized to greater than one independent micro-array were internally normalized to the averaged dye ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), then viral spots were averaged. Each viral dye ratio was normalized to that of the KSHV true late ORF25/Major capsid protein transcript (as quantitated by Northern blotting, the MCP transcript was unaffected by Rta-ER WT and mutants in presence of protein synthesis inhibitors).
The five resulting data sets were tested for significant changes in gene expression using two methods: Method 1. Average number of standard deviations from mean of each data set: dye ratios of each spot for Rta_NLS1,2-ER were divided by dye ratios for ER vector to determine spot-specific effects of 4-OHT addition. Means of the triplicate dye ratios were calculated to determine gene specific effects of 4-OHT addition. Each data set was log transformed and fit to a normal (Gaussian) distribution, and the means of the normalized gene-specific values were calculated. The resulting set of gene-specific means was fit to a normal distribution, outliers were excluded using Peirce’s criterion (hypothesis: one outlier per gene), and the average number of standard deviations from the mean was calculated for each gene. Method 2. q-values: gene specific t tests and q values (chance of being false positive, expressed as a percentage) were calculated by comparing the dye ratios of each spot for Rta_NLS1,2-ER to the dye ratios for ER vector using the software program significance analysis of microarrays (SAM).
Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein
Data table header descriptions
ID_REF
VALUE_median
log (base 2) transform of the ratio of the medians.
VALUE_Linear
inverse log of VALUE
VALUE_GAPDHNorm
VALUE_Linear normalized to average of 3 replicates of VALUE_Linear of GAPDH
VALUE
VALUE_GAPDHnorm normalized to average of 3 replicates of VALUE_Linear of ORF25
X
the X-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Y
the Y-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Dia.
the diameter in µm of the feature-indicator.
F635 Median
median feature pixel intensity at wavelength #1 (635 nm).
F635 Mean
mean feature pixel intensity at wavelength #1 (635 nm).
F635 SD
the standard deviation of the feature pixel intensity at wavelength #1 (635 nm).
F635 CV
the coefficient of variation of the feature pixel intensity at wavelength #1 (635 nm).
B635
total background pixel intensity at wavelength #1 (635 nm).
B635 Median
the median feature background intensity at wavelength #1 (635 nm).
B635 Mean
the mean feature background intensity at wavelength #1 (635 nm).
B635 SD
the standard deviation of the feature background intensity at wavelength #1 (635 nm).
B635 CV
the coefficient of variation of the background pixel intensity at wavelength #1 (635 nm).
% > B635+1SD
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635 nm).
% > B635+2SD
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm).
F635 % Sat.
the percentage of feature pixels at wavelength #1 that are saturated.
F532 Median
median feature pixel intensity at wavelength #2 (532 nm).
F532 Mean
mean feature pixel intensity at wavelength #2 (532 nm).
F532 SD
the standard deviation of the feature intensity at wavelength #2 (532 nm).
F532 CV
the coefficient of variation of the feature pixel intensity at wavelength #2 (532 nm).
B532
total background pixel intensity at wavelength #2 (532 nm).
B532 Median
the median feature background intensity at wavelength #2 (532 nm).
B532 Mean
the mean feature background intensity at wavelength #2 (532 nm).
B532 SD
the standard deviation of the feature background intensity at wavelength #2 (532 nm).
B532 CV
the coefficient of variation of the background pixel intensity at wavelength #2 (532 nm).
% > B532+1SD
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #2 (532 nm).
% > B532+2SD
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm).
F532 % Sat.
the percentage of feature pixels at wavelength #2 that are saturated.
Ratio of Medians (635/532)
the ratio of the median intensities of each feature for each wavelength, with the median background subtracted.
Ratio of Means (635/532)
the ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.
Median of Ratios (635/532)
the median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Mean of Ratios (635/532)
the geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Ratios SD (635/532)
the geometric standard deviation of the pixel intensity ratios.
Rgn Ratio (635/532)
the regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.
Rgn R2 (635/532)
the coefficient of determination for the current regression value.
F Pixels
the total number of feature pixels.
B Pixels
the total number of background pixels.
Circularity
a measure of circularity from 0 to 100, using a metric based on the variance of the distance of each boundary pixel to the centroid of the feature: 100 is most circular, 0 is most non-circular.
Sum of Medians (635/532)
the sum of the median intensities for each wavelength, with the median background subtracted.
Sum of Means (635/532)
the sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.
F635 Median - B635
the median feature pixel intensity at 635 nm. with the median background subtracted.
F532 Median - B532
the median feature pixel intensity at 532 nm. with the median background subtracted.
F635 Mean - B635
the mean feature pixel intensity at 635 nm. with the median background subtracted.
F532 Mean - B532
the mean feature pixel intensity at 532 nm. with the median background subtracted.
F635 Total Intensity
the sum of all pixel intensities in the feature at 635 nm.
F532 Total Intensity
the sum of all pixel intensities in the feature at 532 nm.
SNR 635
the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
SNR 532
the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
Flags
the type of flag associated with a feature.
Normalize
flag column describing if the feature was used to calculate the normalization factors (1 for used, 0 for not used).
Autoflag
reports whether or not a feature has been flagged from the Flag Features dialog box. It applies to good and bad flags only.
