Novel in vivo approaches for manufacturing RNA agents are warranted to complement conventional methods such as in vitro transcription and chemical synthesis. Recently, we have identified and utilized unique tRNA-fused pre-miRNA carriers to establish a robust RNA molecular bioengineering technology that allows one to consistently and efficiently produce target, bioengineered or biologic RNA molecules bearing small interfering RNA (siRNA) payloads (BioRNA/siRNA). Here, we describe a streamlined method with a detailed protocol for the rational design, molecular cloning, heterologous overexpression, and chromatography purification of large quantity (multi-milligrams from 0.25 L of bacterial culture) and high quality (> 98% homogeneity and <5 endotoxin unit/μg RNA) of target BioRNA/siRNA molecules that are ready for basic research as well as experimental therapy.