Starting at one year of age, fed a standard AIN-93G diet, ad libitum.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Qiagen RNAeasy kit
Label
biotin
Label protocol
standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has 24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
Description
Heart 1B SD 27
Data processing
Data was extracted using the Illumina BeadStudio software(v1). Any spots at or below the background were filtered out using an Illumina detection score of 0.98 and below. The natural log of all remaining genes were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores provided as supplementary file.