Male CBK (on the C57Bl/6J background), as well as littermate controls, were housed at the Center for Comparative Medicine at the University of Alabama at Birmingham, under temperature-, humidity-, and light- controlled conditions. A strict 12-hour light/12-hour dark cycle regime was enforced (lights on at 6AM; zeitgeber time [ZT] 0); the light/dark cycle was maintained throughout these studies, facilitating elucidation of the potential roles for the cardiomyocyte circadian clock under physiological conditions (as such, diurnal variations were investigated). Mice received food and water ad libitum.
Extracted molecule
total RNA
Extraction protocol
A standard RNA extraction protocol using Tri-reagent was used.
Label
Cy3
Label protocol
Total RNA was converted to cDNA by reverse transcription using ArrayScript reverse transcriptase and T7-(dT)24 primers, followed by second-strand synthesis to generate double-stranded cDNA (Ambion). After purification, the cDNA was converted to biotin-labeled cRNA, hybridized to the BeadChip (Illumina), and stained with strepavidin-Cy3 for visualization.
Hybridization protocol
See above
Scan protocol
BeadChips were visualized using an iScan system (Illumina, Inc.).
Description
WT- zt3 Ad libitum feeding, standard chow
Data processing
Stata version IC10.0 (Stata Corp., San Antonio, TX) was used to perform two-way ANOVA to investigate main effects of time and/or genotype, followed by Bonferroni post hoc analyses for pair-wise comparisons. Rhythmic parameter comparisons of were calculated using Stata version IC10.0 to perform cosinor analysis. The cosinor analyses were performed based on the assumption that the free-running period is fixed at 24 h. Data was considered rhythmic if the p-value of the R2 of the linearized cosinor function of f(t) = M + Cos(2πt/24) + Sin(2πt/24) was less than 0.05. In all analyses, the null hypothesis of no model effects was rejected at p<0.05.
