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Rock samples intended for molecular analyses were maintained at −80°C until the time of analysis.
Aliquots of the RNA used for the microarray analyses were maintained at -80°C until needed for RT-PCR verification of microarray data.
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Incomplete transcripts without sufficient overlap in alignments and transcripts less than 200 amino acids in length (except for the OBPs where full-length transcripts are generally shorter than 200 amino acids) were excluded from phylogenetic analyses to ensure that the analysed transcripts corresponded to individual genes and that greater accuracy in the analyses was maintained.
Briefly, quality control of the analyses was maintained by analysing a method blank (calf serum) and two spiked calf serum samples (20 ng/ml, all analytes) along with every 17 samples.
For each chronic disease and age sex group, mortality rates were assigned to each level of alcohol consumption such that the RRs from the meta-analyses were maintained and the total risk in the population produced the recorded number of mortalities.
Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes.
Although some domains were collapsed or discarded during the ESEM analyses, most were maintained in the final version of the healthcare team survey.
On the day of birth, all pups were randomized and redistributed to the dams with a litter size of 9 10 to maintain a standard nutritional status; for treatment groups with high pup mortality rates (not used for neuro-chemical analyses), litter sizes were maintained in this range by combining groups of survivors.
Models were as outlined above, with the exception that the binary variable for exceeding alcohol intake guideline ('within guidelines: ≤4 standard alcoholic drinks' versus '≥5 standard alcoholic drinks') in typical and maximum AmED session was included in Step 4. Across all analyses, significance levels were maintained at p < .05.05
In general, observed associations seen in bivariate analyses (chi square) were maintained after adjusting for other independent variables with the exception of age, born in the U.S., poverty status and smoking status for which no associations were seen in the fully adjusted model.
The current analyses assessed whether these effects were maintained at 2-year follow-up.
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