Highly Sensitive and Multiplexed One-Step RT-qPCR for Profiling of Circadian Gene Expression Using Microparticles
- 주제(키워드) Highly Sensitive and Multiplexed One-Step RT-qPCR , Circadian rhythm , PIN(Primer Immobilized Network)
- 발행기관 고려대학교 대학원
- 지도교수 심상준, 김상경
- 발행년도 2019
- 학위수여년월 2019. 8
- 유형 Text
- 학위구분 석사
- 학과 대학원 화공생명공학과
- 원문페이지 70 p
- 실제URI http://www.dcollection.net/handler/korea/000000084499
- UCI I804:11009-000000084499
- DOI 10.23186/korea.000000084499.11009.0000936
- 본문언어 영어
- 제출원본 000045999171
초록/요약
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. I 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 200pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. I 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.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1 Preparation of templates 5
2.1.1 Primer design 5
2.1.2 Synthetic RNA & Synthetic DNA 5
2.1.3 RNA extraction 6
2.1.4 mRNA from Human hair follicle cells 6
2.2 Preparation of encoded PIN particles 7
2.3 Quantitative PCR using microparticles 8
2.3.1 Real-time one-step RT-quantitative PCR with PIN 8
2.3.2 Single cell detection with PIN 9
2.4 Cell based assay 11
2.4.1 Cell culture 11
2.4.2 10uM Forskolin treatment 11
3. Results and discussion 12
3.1 Validation of one-step RT-qPCR for mRNA quantification 12
3.1.1 Validation of PCR performance of thermo-responsive PIN (tPIN) 12
3.1.2 One-step RT-qPCR validation 17
3.1.3 The effects of RNA pre-concentration in PIN particle 24
3.1.4 Particle-based one-step RT-qPCR efficiency and LOD (Limit of Detection) from total cell RNA 28
3.2 mRNA expression level of circadian rhythm from HeLa cells and human hair follicle cells 32
3.2.1 Multiplex one-step RT-qPCR for circadian genes 32
3.2.2 Circadian gene expression of synchronized HeLa cells 36
3.2.3 Circadian gene expression of from human hair follicles 38
3.3 Multiplex quantitative analysis in more limited conditions 43
3.3.1 Single cell detection 43
3.3.2 Snapshots of circadian cycle from 1 hair follicle 46
4. Conclusion 49
References 50
Supplementary Information 56
