Each microfluidic chip contained 350 mature miRNAs of Rat as per Sanger selleckbio miRBase database. Each miRNA was spotted on the array nine times and for each RNA sample two chips were used. There were 16 sets of Inhibitors,Modulators,Libraries control probes on each array. There were 10 positive controls. There were 10 negative controls. The background subtracted signals were used for statistical tests and clustering analysis. Microarray data analysis MiRNA microarray data were analyzed by LC Sciences by subtracting the background and normalizing the signals. Blank spaces represent signal values below detection level. A transcript to be listed as detectable must meet at least two conditions signal intensity higher than 3 and spot CV 0. 5. CV is calculated by.
When repeating probes are present on an array, Inhibitors,Modulators,Libraries a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. The miRNA microarray data used the total gene signal, which was the average value of repeating spots. During data process, bad spots that have signal values deviated more than 50% of average values of repeating spots and or spot CV larger than 0. 5 are discarded. Differentially expressed signals were determine by t test with p 0. 05. Isolation of cardiomyoblasts and simulated ischemia Ventricular cardiomyoblasts were isolated from male Wistar rats by perfusion of hearts with collagenase type II and cultured, as previously described. For this purpose adult male rats were euthanized using deep isoflurane anaesthesia, hearts were rapidly ex cised, washed with ice cold 0.
9% NaCl and connected to the Langendorff perfusion system. Anaesthesia depth was monitored by limb withdrawal using toe pinching. To separate cardiomyoblasts from non cardiac Inhibitors,Modulators,Libraries cells, cardio myoblasts were sedimented by low force and short cen trifugations and finally without centrifugation in medium containing 4% bovine serum albumin. To prevent growth of non myocytes, medium was supplemented with 10 umol L cytosine B d arabino furanoside. After 1 hour of plating, cells were washed with culture medium to remove non attached cells. A high purity of cardiomyoblast culture was confirmed by light microscopy. Third day after preparation cardiomyoblasts were exposed to simulated in vitro ischemia consisting of glucose free anoxia at pH 6. 4 as previously described.
After 3 hours of ischemia, total RNA was isolated and used for further analysis. TaqMan real time microRNA PCR Total RNA was reverse transcribed using the TaqMan miRNA Reverse Transcription Kit and miRNA specific stem loop primers in a small scale RT reaction, and 5. 0 ml of input RNA, components other than the input RNA Inhibitors,Modulators,Libraries were prepared as a larger Inhibitors,Modulators,Libraries volume master mix using a Tetrad2 Peltier Thermal EPZ-5676 Cycler at 16 C for 30 min, 42 C for 30 min and 85 C for 5 min. For miRNAs and snoRNA, 4. 0 ml of RT product was combined with 16. 0 ml of PCR assay reagents to generate a PCR of 20. 0 ul of total volume.