Multichannel Reverse Transcription-Polymerase Chain Reaction Microdevice for Rapid Gene Expression and Biomarker Analysis

Abstract

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.

Integrated microfluidic bioprocessor for single-cell gene expression analysis

“…The first region is a 3-valve pump for moving material from the sample inlet through the reactor region (21). In the reactor region, single cells are captured, lysed, and the mRNA of interest is reverse transcribed and amplified by RT-PCR (22). The affinity capture region comprises a hold chamber that acts as a reservoir and a capture chamber where amplicons are immobilized, purified, and concentrated in an affinity capture gel matrix (23).…”

“…It should also be possible to perform a quantitative single-cell analysis of the effects of siRNA treatment on expression in hESCs when Oct4 mRNA targeting is used to trigger differentiation into trophoblast-like cells (30,31). Moreover, based on our previous detection of Ͻ11 mRNA molecules per reactor (22), our microfluidic device may ultimately enable studies of expression from individual cells at the single-transcript level, once improved product capture, purification, and injection processes are fully enabled and integrated. Overall, our approach offers many exciting prospects for revealing the stochastic variation in gene expression that underlies the ensemble average.…”

“…The fabrication protocol is similar to that used in previous nucleic acid amplification microdevices (22). Briefly, to form the pneumatic manifold wafer, valve seats and actuation channels were photolithographically defined and etched to a depth of 38 m on a 0.5-mm-thick borofloat 100-mm glass wafer.…”

On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets

“…In microfabricated compartments, RT-PCR in 450 pL reactors has been demonstrated down to an estimated level of 34 RNA copies 6 . Nanoliter volume RT-PCR has been integrated to on-chip capillary electrophoresis to detect 11 RNA copies in 45 minutes 7 . RT-PCR has also been performed in batch-generated emulsions at the single copy level 8 .…”

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

“…To more fully realise the potential of microfluidic systems, 10 multiple functions can be combined on one chip. PCR has been integrated in a single microdevice with pre-amplification modules, such as nucleic acid extraction 11 and purification plus pre-concentration, 12 or with post-amplification analytical modules, such as capillary electrophoresis (CE) 13,14 and DNA microarrays. 15 Fully integrated microfluidic PCR devices with pre-and post-amplification modules have also been reported, [15][16][17] and portable versions of fully integrated microfluidic PCR devices with all necessary supporting systems, such as pumps and optical detection, have been achieved.…”