Multiplexed on-chip real-time PCR using hydrogel spot array for microRNA profiling of minimal tissue samples

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>Since microRNAs (miRNAs) have been considered as regulators of messenger RNA (mRNA) translation, the development of the simple, multiplex, and quantitatively precise miRNA profiling techniques becomes more significant. Here, an on-chip multiplex real-time quantitative polymerase chain reaction (qPCR) for miRNA profiling is demonstrated with a primer-immobilized network (PIN) array, which consists of hundreds of hydrogel spots. The array renders highly dense reaction cells of 20 × 20 in 1 cm<SUP>2</SUP>. Uniform performance of the 400 real-time PCR reservoirs was achieved through our fabrication process. The amplicons and their fluorescent signals were isolated in each hydrogel spot, whose detection limit was measured to 16 aM, covering a seven-log concentration range. With the PIN spots of different primers, multiple miRNAs could be quantified in a single reaction out of very limited amount of RNA. For proof of concept, ten different miRNAs from the medial habenula of a mouse which is small region of the brain were successfully analyzed.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We achieved 400 hydrogel spots array in 1 cm<SUP>2</SUP> with excellent structural and functional uniformity. </LI> <LI> We developed the multiplex qPCR array chip for fast and simple workflow. </LI> <LI> This qPCR chip successfully detected as low as 16 aM of miRNA with a wide dynamic range of seven logs. </LI> <LI> Ten miRNAs from medial habenula of mouse brain were profiled at once in a 10 × 10 array. </LI> </UL> </P>