Cells (70-80% confluent) cultured in 75 cm2 flasks were harvested using 1 ml/10 cm2 of TRIzol reagent (Invitrogen) and stored at -80ºC until use. RNA and DNA were extracted using TRIzol reagent and according to the manufacturers protocols. Nucleic acids were quantified using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA) and quality was visually confirmed by gel electrophoresis.
Label
Cy3
Label protocol
cDNA probes were generated from 30μg of total RNA, with an oligo(dT) [(dT)20-VN] primer (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with incorporation of dUTP, and indirectly labeled with cyanine-5 dCTP and cyanine-3 dCTP (Perkin-Elmer). cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands).
Human breast cancer cell line resistant to Vitamin D
Extracted molecule
total RNA
Extraction protocol
Cells (70-80% confluent) cultured in 75 cm2 flasks were harvested using 1 ml/10 cm2 of TRIzol reagent (Invitrogen) and stored at -80ºC until use. RNA and DNA were extracted using TRIzol reagent and according to the manufacturers protocols. Nucleic acids were quantified using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA) and quality was visually confirmed by gel electrophoresis.
Label
Cy5
Label protocol
cDNA probes were generated from 30μg of total RNA, with an oligo(dT) [(dT)20-VN] primer (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with incorporation of dUTP, and indirectly labeled with cyanine-5 dCTP and cyanine-3 dCTP (Perkin-Elmer). cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands).
Hybridization protocol
cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands) and mixed with 12 µg poly(dA) (Amersham), 60 µg yeast tRNA (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands), and 24 µg Cot-1 DNA (Invitrogen). The labeled target was dissolved in 127-µL hybridization mixture containing 46% formamide (Invitrogen), 9.5% dextran sulfate (U.S. Biochemical Corp., Cleveland, USA), 2x SSC, and 0.2% SDS. The labeled target was heated to 70°C for 10 minutes and annealed at 37°C for 1 hour. Slides were hybridized at 37°C overnight (14h). After hybridization, the slides were washed in the HybArray 12, with 50% formamide (Fluka, Sigma-Aldrich Chemie), 2x SSC (pH 7) at 35°C for 15 minutes followed by PI buffer (0.1 mol/L sodium phosphate, 0.1% Igepal Ca630 (pH 8)) at room temperature and three washes of 0.2x SSC, 0.1x SSC, and 0.01x SSC at room temperature followed by centrifugation.
Spot analysis and quality control were fully automated using BlueFuse version 3.2 (BlueGnome, Cambridge, UK) and spots with quality flag <1 or confidence value <0.1 were excluded from further analysis. After scanning and data acquisition, genes with spots flagged in more than one array were excluded from further analysis. For the remaining genes, the value of a flagged spot was imputed using K nearest neighbors’ imputation as implemented in the R-package “impute”. Curvature in the MA plots was limited but nevertheless normalized using lowess as implemented in the R-package “MAAnova”.
Understanding Vitamin D resistance using expression microarrays
Data table header descriptions
ID_REF
AMPL_CY3
Total signal in channel 1 (Cy3)
AMPL_CY5
Total signal in channel 2 (Cy5)
FLAG
0-value not flagged, 1-value flagged in one array (IMPUTED), 2-value flagged in more than one array (NOT USED), 3-value flagged due to quality/confidence (NOT USED)
IMPUTE_CY3
Cy3 imputed value using the R-package “impute”
IMPUTE_CY5
Cy5 imputed value using the R-package “impute”
LOWESS_CY3
Lowess normalized Cy3 value
LOWESS_CY5
Lowess normalized Cy5 value
VALUE
-[INV_VALUE]
INV_VALUE
Log, base 2, of the ratio of the Lowess normalized Cy3 value divided by Lowess normalized Cy5 value
