Total rna isolation kits

U46619, SQ29548, and 2-APB were purchased from Cayman Chemical (Ann Arbor, MI). Hanks balanced salt solution (HBSS) and Fura-2 AM were obtained from Invitrogen (Carlsbad, CA). Enzymes for cardiomyocyte digestion were purchased from Worthington Biochemical (Lakewood, NJ). Total RNA Isolation kits were purchased from IBI Scientific (Peosta, IA), and the real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using a TaqMan RNA-to-CT 1 step kit and primers and probes from ABI (Carlsbad, CA). Primary antibody for TXA2R was purchased from Abcam (Cambridge, MA). Gentamicin and fetal bovine serum was obtained from Sigma-Aldrich (St. Louis, MO). DeadEnd Fluorometeric TUNEL stain was purchased from Promega (Madison, WI). All remaining reagents were purchased from Fisher Scientific.

Total RNA was isolated from Dmp1 null and WT mouse ventricles using the Total RNA isolation kits from IBI Scientific (Peosta, IA). One step real-time RT-PCR was performed as previously described (Wacker and Godard, 2005 (link)) using the TaqMan RNA-to-CT 1 step kit from Applied Biosystems (Carlsbad, CA). Premade TaqMan primer/probes for genes of interest as well as the housekeeping gene, β-actin, were ordered from Applied Biosystems. Genes involved in cardiac hypertrophy or heart failure which included atrial naturietic peptide (ANP, Mm01255747_g1), skeletal muscle α actin (SkAct, Mm00808218_g1), β-myosin heavy chain (βMHC, Mm00600555_m1), α-myosin heavy chain (αMHC, Mm00440359_m1), sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2a2, Mm01201431_m1) as well as genes involved in cardiac remodeling, vimentin (Mm01333430_m1), and collagen 1a1 (Mm00801666_g1), were tested. Relative gene expression was quantified by 2^−ΔCT calculations utilizing β-actin as the housekeeping gene. Fold change was then calculated by dividing Dmp1 null by WT expression and the results were averaged. One fold change equal expression to the WT and is indicated by a dashed line in Figure 4. β-actin was chosen as a housekeeping gene as Dmp1 null and WT had minimal differences in β-actin gene expression.