Project researchers envision that these stable markers will be used in casework to increase the precision of age estimates of entomological evidence. A description of study methods addresses colony care, the sampling scheme, miRNA extractions and sequencing, sequence analysis, qPCR, protein extractions, DIGE, Western Blot, and the detection of miRNA when traditionally preserved. Data analysis is described. The project has identified just over 200 miRNAs present in the development of three species of flies that have forensic relevance, with flesh fly data forthcoming. Researchers found that expression of these miRNA is differential through intrapuparial development and that statistical analysis of these expression patterns can be used to differentiate the amount of time that has passed since pupariation began. Project findings suggest there are stable and conserved molecular markers for determining the age of pupae and differentiating feeding and post-feeding third instar larvae. In addition, researchers optimized a sex identification assay for three species of carrion flies. Also, significant differences were found in the development of B. plinthopyga on various substrates, which is a step toward increased precision for estimates of this species, which has been found in many indoor cases in Houston, TX.