Highly sensitive and multiplexed one-step RT-qPCR for profiling genes involved in the circadian rhythm using microparticles

Abstract

Abstract Given the growing interest in molecular diagnosis, highly extensive and selective detection of genetic targets from a very limited amount of samples is in high demand. We demonstrated the highly sensitive and multiplexed one-step RT-qPCR platform for RNA analysis using microparticles as individual reactors. Those particles are equipped with a controlled release system of thermo-responsive materials, and are able to capture RNA targets inside. The particle-based assay can successfully quantify multiple target RNAs from only 200 pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. We carried out 8-plex one-step RT-qPCR using tens of microparticles, which allowed extensive mRNA profiling. The circadian cycles were shown by the multiplex one-step RT-qPCR in human cell and human hair follicles. Reliable 24-plex one-step RT-qPCR was developed using a single operation in a PCR chip without any loss of performance (i.e., selectivity and sensitivity), even from a single hair. Many other disease-related transcripts can be monitored using this versatile platform. It can also be used non–invasively for samples obtained in clinics.