Brain homogenates (10% wt/vol for murine samples and 20% wt/vol for ruminant controls) were prepared by using ribosylation tubes (Bio-Rad Laboratories, HerCAles, CA, USA).
Although this approach has not been used much for murine samples, this strategy has already been adopted for reducing errors in the analysis of human and veterinary samples and has been used as an internal quality control for both a variety of samples and types of analyses [ 15– 17].
RNA was extracted, amplified and assessed for quality as described for murine samples.
This is likely explained by the amount of HS injected onto the LC/MS system (higher for bovine samples than for murine samples, according to total ion counts) and the intensity of the tetrasaccharides was proportional to the overall signal for all disaccharides observed in the LC/MS datasets.
Notably for miRNA studies of cardiac tissue, more information is available for murine samples, including studies on knockout mice.
For murine samples, triplicate reactions of three biological replicates were utilized.
cRNA was hybridized against the HG-FOCUS array (Affymetrix, >8700 probe sets encoding ∼8400 genes) and imaged as described for murine samples.
Briefly, sections were blocked in either normal goat, rabbit or horse serum containing 10%(v/v) from stock avidin solution (Vector Labs) for 20 min followed by 1 hr incubation with anti-PPARβ/δ (R&D, PP-K9436) at 1∶100 overnight at 4°C for human samples (incubation for 1 h at room temperature yielded comparable results)., and 1∶1000 for murine samples.
For detection of HIF-1α isoforms, a monoclonal mouse anti-human HIF-1α antibody was used for patient samples (α67; Novus Biologicals) and polyclonal rabbit anti-mouse HIF-1α antibodies were used for murine samples (PM16, obtained from a rabbit immunized with a peptide containing amino acids 553 to 669 of mouse HIF-1α HIF-1αdly gifriendlyPatrick Maxwell, Imperial Hospital, London, UK).
