strain: 129S1/Sv1mJ age: Embryonal Day 10 tissue: cardiac tissue
Treatment protocol
The presence of a plug was indicative of day 0 pregnancy. At this time, each folate group was divided in two sub-groups of control (CTL) and trichloroethylene exposed mice (TCE), and maternal exposure to 10ppb TCE was started via drinking water.
Growth protocol
129S1/Sv1mJ mice were obtained from Jackson Laboratories. Female mice were assigned to 3 different folate diets for four weeks before mating: 0, 2, or 8mg/kg folate (Dyets Inc), and received water ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from pooled embryo hearts (n~ 35, corresponding to 4-6 litters) and purified using Trizol /Rneasy Mini Kits (Qiagen, Valencia, CA)
Label
biotin
Label protocol
The arrays were processed according to the protocol (GeneChip Whole Transcript(WT) Sense Target Labeling Assay Manual, version4) established by Affymetrix. Briefly, 400ng of total RNA were reverse transcribed into double stranded cDNA by performing a two strand synthesis using the Affymetrix WT cDNA Synthesis and Amplification kit. The entire cDNA was then in vitro transcribed overnight and cRNA cleaned using the Affymetrix GeneChip Sample Cleanup Module and quantified on a NanoDrop 1000(Thermo Scientific, Wilmington, DE). Then, 8ug of cleaned-cRNA was reverse transcribed into ssDNA, which was used as template for the labeling reaction.
Hybridization protocol
An aliquot of the labeled DNA was combined with the hybridization solution, hybridized overnight on the Mouse Gene 1.0ST array in an Affymetrix GeneChip Hybridization Oven 640.
Scan protocol
Once the wash and stain procedure was completed, the arrays were scanned using the Affymetrix GeneChip Scanner 3000 with 7G upgrade.
Description
Total RNA was extracted from 35 pooled cardiac tissues pooled from 4-6 different litters. Dams were kept on a diet containing 2mg/kg folate. Two replicates of each samples were used for each hybridization
Data processing
The image generated was analyzed using the Affymetrix GeneChip Operating Software (GCOS) were .DAT (image), .CEL (cell intensity data), .CHP (probe analysis data) files were generated.The Affymetrix mouse gene ST arrays were processed with a bioconductor library (http://www.bioconductor.org) designed for this array. The arrays were then normalized using the RMA function in the oligo library of bioconductor. In order to identify poor quality arrays, Spearman correlation coefficients between all arrays were computed. A plot of the coefficients revealed that correlations between the arrays were 0.98 or greater, indicating that a relatively small number of genes were changing in response to the treatments. As a further control, the distribution of values for positive and negative control probes were examined on each array and found to have the correct expected separation. Since there were only two biological replicates due to the complexity of the sample preparation, a specific approach was used that identified probes that showed a consistent change between biological duplicates. A cutoff value for the change between treatments of 1.5 fold was chosen based on an examination of the distribution of expression values on these arrays and identified between 200-1000 probes that were varying to the greatest extent. Probe lists were generated as described below. Briefly, the objective was to find genes that were changing between treatments while reducing the effects of the biological variation between duplicate samples. To compare two treatments, probe expression ratios for all 4 possible combinations of the duplicate arrays were calculated. If 2 or more of these ratios were 1.5 fold or greater, the gene was considered to be changed.
