tissue: Cerebral cortex gender: male age: 3-weeks-old
Extracted molecule
total RNA
Extraction protocol
RNA was prepared from each fresh frozen tissue using NucleoSpin RNA II kit (TaKaRa-Bio, Japan) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Total RNA was amplified and labeled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, Palo Alto, CA) following the manufacturer's instructions. Briefly, 100ng of total RNA was reversed transcribed to double-strand cDNA using a poly dT-T7 promoter primer. Primer, template RNA and quality-control transcripts of known concentration and quality were first denatured at 65 ℃ for 10 min and incubated for 2 hours at 40 ℃ with 5X first strand Buffer, 0.1 M DTT, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was inactivated at 70 ℃ for 15 min. cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40 ℃ for 2 hours. Labeled cRNAs were purified using QIAGEN’s RNeasy mini spin columns and eluted in 30 μl of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.
Hybridization protocol
For each hybridization, 1.65 μg of Cy3 labeled cDNA were hybridized at 65 ℃ for 17 hours to an Agilent Mouse GE 8x60K Microarray (Design ID: 028005).
Scan protocol
Microarrays were scanned using an Agilent Technologies Scanner G2505C.
Description
Bisphenol A (BPA) was exposed maternally and via lactation
Data processing
Intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. We only used features which were flagged as no errors (present flags) and excluded features which were not positive, not significant, not uniform, not above background, saturated and population outliers (marginal and absent flags). Normalization was performed using Agilent GeneSpring GX version 11.0.2. (per chip: normalization to 75 percentile shift; per gene: normalization to median of all samples).
