Title Page
Contents
ABSTRACT 7
Ⅰ. Introduction 9
Ⅱ. Material and Methods 11
2.1. In vitro transcription of RNA 11
2.2. Calculation of the N/P ratio 11
2.3. Purification of IVT RNA using mesoporous silica-based chromatography 12
2.4. Spermidine-conjugated mesoporous silica for purification of IVT RNA 13
2.5. Cellulose-based purification of IVT RNA 14
2.6. RiboGreen assay for RNA quantification 14
2.7. Quantification of dsRNA by ELISA 15
2.8. Dot blotting analysis for dsRNA detection 15
Ⅲ. Results and discussion 17
3.1. Adsorption of IVT RNA to silica in the presence of spermidine 17
3.2. Characteristics of RNA purification with mesoporous silica-based spin column chromatography 20
3.3. Elution reagents and polyamines for RNA purification with mesoporous silica-based chromatography 22
3.4. Mesoporous silica rather than spherical silica as facile adsorption/desorption of RNA 25
3.5. Evaluation of the elution reagents for recovery of mesoporous silica-adsorbed RNA 27
3.6. Removal of dsRNA from IVT RNA using mesoporous silica-based spin column chromatography 30
Ⅳ. Conclusions 33
References 34
Abstract (in Korean) 37
Figure 1. Spermidine in the in vitro transcription buffer promotes RNA binding to mesoporous silica. The LiCl-precipitated IVT RNA (2 μg) mixed with mesoporous silica... 19
Figure 2. Assessment of mesoporous silica-based spin column chromatography for IVT RNA (A) Experimental process for determining the adsorption/desorption of IVT RNA... 21
Figure 3. Evaluation of mesoporous silica-based spin column chromatography; elution reagents, and polyamines for purification of IVT RNA (A) Desorption of RNA from... 24
Figure 4. Examination of two types of silica particles as RNA adsorbent; mesoporous silica and spherical silica. The LiCl-precipitated IVT RNA (2 μg) mixed with spermidine... 26
Figure 5. Evaluation of the elution reagents for recovery of silica-adsorbed RNA. The LiCl-precipitated IVT RNA (10 μg) was purified by mesoporous silica-based spin... 29
Figure 6. Removal of dsRNA from IVT RNA by mesoporous silica-based spin column chromatography. The unpurified IVT RNA (10 μg) was purified by mesoporous silica-... 32