Microfluidics　or　Bio-MEMS　technology　offers　significant　advantages　for　performing　high-throughput　screens　and　sensitive　assays.　The　ability　to　correlate　single-cell　genetic　information　with　cellular　phenotypes　is　of　great　importance　to　biology　and　medicine　because　it　holds　the　potential　to　gain　insight　into　disease　pathways　that　is　unavailable　from　ensemble　measurements.　Previously,　we　reported　two　kinds　of　prototypes　for　integrated　on-chip　gene　expression　profiling　at　the　single-cell　level,　and　the　throughput　was　designed　to　be　6.　In　this　work,　we　present　a　five-layer　microfluidic　system　for　parallelized,　rapid,　quantitative　analysis　of　RNA　templates　with　low　abundance　at　the　single-cell　level.　The　microchip　contains　two　multiplexors　and　one　partitioning　valve　group,　and　it　leverages　a　matrix　(6　x　8)　of　quantitative　reverse　transcription　polymerase　chain　reaction　(RT-qPCR)　units　formed　by　a　set　of　parallel　microchannels　concurrently　controlled　by　elastomeric　pneumatic　valves,　thereby　enabling　parallelized　handling　and　processing　of　biomolecules　in　a　simplified　operation　procedure.　A　comprehensive　metallic　nanofilm　with　passivation　layer　is　used　to　run　polymerase　chain　reaction　(PCR)　temperature　cycles.　To　demonstrate　the　utility　of　the　approach,　artificial　synthesized　RNA　templates　(XenoRNA)　and　mRNA　templates　from　single　cells　are　employed　to　perform　the　48-readout　RT-qPCRs.　The　PCR　products　are　imaged　on　a　fluorescence　microscope　using　a　hydrolysis　probe/primer　set　(TaqMan).　Fluorescent　intensities　of　passive　reference　dye　and　a　fluorescein　amidite　reporter　dye　are　acquired　and　measured　at　the　end　of　PCR　cycles.　(C)　2016　Elsevier　Inc.　All　rights　reserved.