Total RNA was extracted from adult hearts using the miRNeasy protocol (Qiagen).
Total Prep RNA Amplification Kit (Illumina).
To further define the physiologic role of miR-22 in the heart we engineered transgenic mice that expressed miR-22 under the control of the myocyte-specific Myh6 promoter (miR-22-Tg). Two independent transgenic mouse lines (low, high) were established that expressed miR-22 approximately 3- and 9-fold higher respectively than WT littermates at 5-weeks of age. Since the level of overexpression in the miR-22-Tg high line was comparable to that observed in human failing hearts, we focused our studies on this line.
Beadlevel data was processed using the beadarray and lumi packages in R and transformed using the variance-stabilizing transformation (VST).
Beadlevel data was processed using the beadarray and lumi packages in R and normalized using the robust spline method (RSN).
Chips were washed, detected and scanned according to the manufacturers instructions.
1500ng of biotinylated RNA was hybridised to Illumina Mouse-6 v1.1 Expression Beadchips overnight at 58¼C.