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

Mi Yeon Kim,Sang Kyung Kim,Junsun Kim,Sang Jun Sim,Heon Jeong Lee,Seungwon Jung,Seunghwa Jeong

doi:10.1038/s41598-021-85728-y

Mi Yeon Kim, Sang Kyung Kim + Show 5 more

Open Access

https://doi.org/10.1038/s41598-021-85728-y

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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.

Highlights

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 examined the efficiency of the PCR amplification using thermo-responsive PIN (tPIN) particles of high porosity for mRNA delivery
We demonstrated the multiplexed one-step RT-qPCR platform using tens of tPIN microparticles

Summary

Introduction

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. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is the gold standard technique for analyzing the mRNA of a specific g ene[1,2] It is widely used in clinics for infectious d iseases[3,4] and chronic diseases such as c ancer[5,6] and metabolic syndromes[7]. This process allows analyzing a number of the genes from a single sample, it involves complicated hands-on operations (such as splitting the samples and filling different reagents into each reaction). It may suffer from contamination issues[15]. Another weakness of this classic approach is the non-target signal, because universal RT primers (such as poly T or hexamers) propagate irrelevant products besides target cDNA16

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