RNA was extracted from 10 mL AF with 30 mL TRIzol LS Reagent (Invitrogen, Carlsbad, CA) and 8 mL chloroform. After RNA extraction, RNA was purified and DNA was removed using the RNeasy® Maxi Kit, including the DNase step according to the manufacturer’s protocol (QIAGEN, Valencia, CA). RNA was precipitated using 3M NaOAc and 100% ethanol, and 80% ethanol was added after 4 hours incubation at -20 °C. cDNA, synthesized from extracted RNA, was amplified and purified using the WT-Ovation™ Pico RNA Amplification System (NuGEN, San Carlos, CA) and the DNA Clean & Concentrator™-25 (Zymo Research, Orange, CA).
Label
biotin
Label protocol
Samples were labeled using the FL-Ovation™ cDNA Biotin Module V2 (NuGEN, San Carlos, CA).
Hybridization protocol
Arrays were washed and stained with streptavidin-phycoerythrin.
Scan protocol
Arrays were scanned on a GeneArray Scanner, using the GeneChip Microarray Suite 5.0 (Affymetrix, Santa Clara, CA).
Description
gene expression data from second trimester fetus with MMC
Data processing
Normalization was performed with the threestep command from the AffyPLM package in BioConductor, using ideal-mismatch background/signal adjustment, quantile normalization, and the Tukey biweight summary method. Identification of individual differentially-expressed genes was performed via two-sided, paired t-tests using the multtest package in BioConductor, with the Benjamini-Hochberg adjustment for multiple testing.
