ABSTRACT This equipment supplement requests funds to purchase an Applied Biosystems QuantStudio 3 Real-Time PCR System, which is critical for completing the aims of the parent grant (NIH MERIT award to Sanford I. Bernstein entitled Genetics and Molecular Biology of Striated Muscle Myosin, GM032443). This instrument will replace an unserviceable Applied Biosystems model 7000, which is approximately 20 years old and is no longer being calibrated or repaired by the vendor. Our current instrument is providing inconsistent results and has shut down prematurely during operation. The QuantStudio 3 Real-Time PCR System will be essential for completing our study on roles of aging-related aggregating proteins in myosin-based skeletal muscle diseases. In this aim, we are assessing the functions of proteins that appear at abnormal levels in aggregates affiliated with Drosophila models of inclusion body myopathy type 3 or myosin storage myopathy. This is accomplished by knocking down (RNAi) or over-expressing (UAS-Gal4 transgenesis) each of these identified proteins in wild-type adults and assessing muscle structure and function. This transcript manipulation approach is then used in the disease model backgrounds to assess possible improvement in muscle phenotypes. For these studies, it is essential to employ real-time quantitative PCR to determine the levels of over- or under-expression of each abnormally expressed gene in both the disease models and in the up- or down-regulated backgrounds. This approach is also useful in assessing transgene expression for our other two grant aims, where we are transgenically increasing levels of the FOXO transcription factor (which improves proteostasis by enhancing clearance of aggregates), to assess whether structural and functional improvements occur in a) skeletal muscles that contain aging-related post-translational myosin modification mimics or b) cardiac muscles that serve as models of dilated cardiomyopathy. The QuantStudio PCR System is advantageous compared to our current outmoded and poorly functional instrument in that it requires less frequent calibration, has VeriFlex temperature control technology to optimize PCR conditions, has better detection capability and resolution, and has onboard and cloud data storage. In addition to these instrument advantages, we will be moving from SYBR Green to TaqMan qPCR assays, wherein we will not simply detect amplified double-stranded DNA (including non-specific amplifications) but will only detect gene-specific amplification. The TaqMan system also has the availability of a library of over 40,000 verified Drosophila primers/probes and the ability to perform multiplex amplification of both the experimental and control transcripts in a single tube, thereby enhancing efficiency of probe design and reproducibility of experimental conditions. Overall, the QuantStudio 3 Real-Time PCR System will provide efficiency, reliability and rigor that is lacking with our outmoded and unrel...