normalization data transformation protocol
Post-processing steps included intra-array loess and inter-array quantile normalization to correct for potential bias resulting from differential stability of cyanine dyes (Limma version 2.12 in Bioconductor 2.12 environment). ID_REF = VALUE = Quantile normalized log2 ratios (test/reference).
array scanning protocol
Microarray slides were scanned using GenePix 4100A microarray scanner.
Hybridization of labeled samples was performed according to manufacturer's instructions (Agilent Low Input Quick Amp Labeling protocol).
Labeling of RNA samples was performed according to manufacturer's instructions (Agilent Low Input Quick Amp Labeling protocol).
nucleic acid extraction protocol
Isolation of RNA from cultured amniocytes was performed using Fujifilm QuickGene-810 automated isolation system (Fujifilm Life Sciences, Tokyo, Japan), using columns in the Fujifilm RNA Cultured Cell kit to capture purified RNA samples. The purity and yield of isolated RNA samples was determined using NanoDrop 2000c spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Amniotic fluid samples were collected between 16th and 18th week of gestation for routine cytogenetic analysis. Primary cell cultures of amniotic fluid were performed according to standard protocols using tissue culture flasks (TPP, Switzerland). Cell cultures were grown in Amnio Max C100 Basal Medium and Amnio Max C100 Supplement (Invitrogen, CA, USA) at 37°C in 5% CO2 environment.