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
EuploidFemale2: gene expression data from normal second trimester fetus
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.
