A microdevice is developed for RNA analysis that integrates one-step reverse transcription and 30 cycles of PCR (RT-PCR) amplification with capillary electrophoresis (CE) separation and fluorescence detection of the amplicons. The four-layer glass-PDMS-glass-glass hybrid microdevice integrates microvalves, on-chip heaters and temperature sensors, nanoliter reaction chambers (380 nL), and 5-cm-long CE separation channels. The direct integration of these processes results in attomolar detection sensitivity (<11 template RNA molecules or approximately 0.1 cellular equiv) and rapid 45-min analysis, while minimizing sample waste and eliminating contamination. Size-based electrophoretic product analysis provides definitive amplicon-size verification and multiplex analysis. Multiplexed differential gene expression analysis is demonstrated on mdh and gyrB E. coli transcripts. RNA splice variant analysis of the RBBP8 gene is used to identify tumorigenic tissue. RT-PCR microdevice analysis of normal breast tissue RNA generates the expected 202-bp normal splice isoform; tumor breast tissue RNA samples generate a 151-bp amplicon signifying the presence of the tumorigenic splice variant. The ability to perform RNA transcript and splice variant biomarker analysis establishes our RT-PCR microdevice as a versatile gene expression platform.