Cows were allocated to one of four treatments; twice-daily milking and adequately-fed (2XAF), 2X and under-fed (fed 60% of requirements; 2XUF), once-daily milking AF (1XAF) or 1XUF at approx wk 5 postpartum. Treatments lastest for 3 weeks and liver tissue was then biopsied.
Extracted molecule
total RNA
Extraction protocol
Liver tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using a TissueLyser instrument (QIAGEN).
Label
Cy5
Label protocol
150ng of total RNA was labelled with Cy5 using an Agilent Low Input Quick Amp (LIQA) kit for each experimental sample, and labelled cRNA was then purified on column, quantified and dye coupling efficiency measured using the Nanodrop (Nanodrop Technologies, Wilmington, DE) instrument. Equimolar amounts of each sample were pooled to generate a reference which was labelled with Cy3 as above.
Cows were allocated to one of four treatments; twice-daily milking and adequately-fed (2XAF), 2X and under-fed (fed 60% of requirements; 2XUF), once-daily milking AF (1XAF) or 1XUF at approx wk 5 postpartum. Treatments lastest for 3 weeks and liver tissue was then biopsied.
Extracted molecule
total RNA
Extraction protocol
Liver tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using a TissueLyser instrument (QIAGEN).
Label
Cy3
Label protocol
150ng of total RNA was labelled with Cy5 using an Agilent Low Input Quick Amp (LIQA) kit for each experimental sample, and labelled cRNA was then purified on column, quantified and dye coupling efficiency measured using the Nanodrop (Nanodrop Technologies, Wilmington, DE) instrument. Equimolar amounts of each sample were pooled to generate a reference which was labelled with Cy3 as above.
Hybridization protocol
825ng of both Cy3 and Cy5 labelled cRNA was used for microarray hybridisation using the Agilent Gene Expression Hybridisation Kit (60-mer oligo microarray protocol version 4.0) (Agilent Technologies, Bioresearch Solutions Unit, 3500 Deer Creek Road, Palo Alto, CA 94034, USA). Dye-labelled, fragmented cRNA was then added to each Agilent 44k 60-mer oligonucleotide microarray, hybridised over night (17 hours), washed and allowed to air dry.
Scan protocol
Arrays were scanned using the Agilent SureScan microarray scanner at 5uM resolution.
Description
Biological Replicate 6 of 10; 2XUF
Data processing
Agilent feature extraction software version 10.1 was used to analyse the scanned Agilent microarray. The 45 scanned microarray image files were uploaded to the feature extraction software. Using the design file (015354) the feature extraction software locates features and converts the extracted data from each feature into a quantitative log ratio. The software removes pixel outliers, does statistics on the non outlier pixels, subtracts background from features and flags any outlier features. The software was then used to perform a LOWESS (locally weighted linear regression analysis) dye normalisation and to calculate a p-value for each feature. Microarray data were imported into Genespring GX 12.5 (Agilent, Palo Alto, CA, USA) and filtered by presence and raw intensity (20th - 100th percentile) before generating a gene level experiment that averaged the expression of probes annotated to the same gene which was then exported for analysis.
