Phenotypic MicroRNA Microarrays
Abstract
:1. Introduction
2. Experimental Methods
2.1. Chemicals and Cell Culture
2.2. miRNA and siRNA Transient Transfection
2.3. Phenotypic NF-κB Assay and Data Analysis
2.4. RNA Extraction and Real-Time PCR
2.5. siRNA and miRNA Printing
2.6. Microarray-Based Phenotypic Assay and Data Analysis
3. Results and Discussion
3.1. Phenotypic Assay of siRNA and miRNA Knock-down Effect in Cellular Model
3.2. RNA Interference Microarrays
3.2.1. High-Density Spot Microarrays for Reverse Transfection
Component of mixture for reverse transfection | |||||
---|---|---|---|---|---|
in wells | on array | ||||
1 | lipofectamine | 0.1 μL | 1 | lipofectamine | 3 μL |
DMEM | 9.9 uL | ||||
2 | SiRNA (1 μM) | 5 μL (1 μM) | 2 | SiRNA (20 μM) | 2 μL (20 μM) |
DMEM | 5 μL | 3 | 0.3 M Sucrose in OptiMEM | 2 μL (0.3 M) | |
RNAse free water | 2 μL | ||||
Mixed and incubate for 20 min | Mixed and incubate for 20 min | ||||
3 | OptiMEM | 80 μL | 4 | siGLO (20 μM) | 2 μL |
5 | 0.2% gelatine | 5 μL | |||
add 100 μL to cells in 1 well/96 well plate or 20 μL in 1 well/384 well plate | Printed 3–6 μL/300 μm spot |
3.2.2. Optimization of the Transfection Reaction for siRNA and miRNA on Microarrays
3.3. Phenotypic Assay for Cellular Monolayer Microarrays with miRNA and siRNA
3.4. Discussion
4. Conclusions
Acknowledgments
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Kwon, Y.-J.; Heo, J.Y.; Kim, H.C.; Kim, J.Y.; Liuzzi, M.; Soloveva, V. Phenotypic MicroRNA Microarrays. Microarrays 2013, 2, 63-80. https://doi.org/10.3390/microarrays2020063
Kwon Y-J, Heo JY, Kim HC, Kim JY, Liuzzi M, Soloveva V. Phenotypic MicroRNA Microarrays. Microarrays. 2013; 2(2):63-80. https://doi.org/10.3390/microarrays2020063
Chicago/Turabian StyleKwon, Yong-Jun, Jin Yeong Heo, Hi Chul Kim, Jin Yeop Kim, Michel Liuzzi, and Veronica Soloveva. 2013. "Phenotypic MicroRNA Microarrays" Microarrays 2, no. 2: 63-80. https://doi.org/10.3390/microarrays2020063