normalization data transformation protocol
Agilent Feature Extraction software (version 10.7.3.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 4 out of 12 have flags in Detected (“ue”or further data analysis. Differentially expressed genes were identified through volcano plot filtering. Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 11.5.1). GO analysis and Pathway analysis were performed in the standard enrichment computation method. ID_REF = VALUE = Normalized signal intensity
array scanning protocol
scanned with using the Agilent DNA Microarray Scanner (part number G2505B)
100μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505B).
1 μg of total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The Labeled cRNAs were purified by RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA
To avoid artifacts from effects of labor, only placental samples obtained from women who had not undergone labor were selected. All samples were collected immediately after Caesarean sections, cut near the center zone of maternal surface villous lobule after deciduas and amnionic membranes were removed, and then rinsed with saline to remove maternal blood.To reduce variations among different individuals, same amount of RNA samples from 5 individuals were pooled for one array.
nucleic acid extraction protocol
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis