Title Page
Contents
ABSTRACT 8
1.1. Introduction 10
2.1. Material and methods 13
2.1.1. Materials 13
2.1.2. Preparing the dumbbell probe 15
2.1.3. miRNA detection using RCT 15
3.1. Results and Discussion 17
3.1.1. Design and construction of DP 17
3.1.2. Feasibility of miRNA Detection 20
3.1.3. Dumbbell probe design and non-specific transcription 22
3.1.4. Optimization condition of DP-RCT 27
3.1.5. Evaluation of miRNA 141 detection system performance 30
3.1.6. Real-sample test of miRNA 141 detection system 34
4.1. Conclusion 37
References 38
국문초록 43
Table 1-1. Oligonucleotide sequences were used in this work. 14
Table 1-2. Comparison of different methods. 33
Table 1-3. Determination of miRNA 141 in 1% human serum samples 35
Figure 1-1. Schematic illustration of the Rolling circle transcription with dumbbell probe and light-up aptamer. 19
Figure 1-2. Feasibility of the miRNA detection based on DP-RCT. Various combinations of reaction components were tested to confirm the fluorescence... 21
Figure 1-3. The nucleic acid structures of the various ODPs and IDPs created using the NUPACK nucleic acid modeling tool. The red bold line indicates the... 24
Figure 1-4. Optimization of the DP structure and T7p length. The relative fluorescence intensity is defined as F/F₀, where F and F₀ indicate the... 25
Figure 1-5. Comparison of non-specific transcription inhibition in RCT as a function of AT probe concentration. The fluorescence intensity of the To1-biotin... 26
Figure 1-6. The Optimization of the concentration of components of DP-RCT. Components refer to rNTP, T7 RNA Polymerase, T7p (T7 promoter), and DP... 28
Figure 1-7. Optimization The optimization of reaction condition on (a) reaction time and (b) reaction temperature for DP-RCT optimization of the transcription... 29
Figure 1-8. (a) Evaluation of miRNA detection specificity using DP-RCT. Fluorescence intensity was assessed in samples containing miRNA 141 (50 nM)... 32
Figure 1-9. The fluorescence intensity as a function of miR-141 concentrations in 1% human serum. The relationship between the fluorescence intensity at 535... 36