In situ visualization of gene expression using polymer-coated quantum-dot-DNA conjugates.
Imaging of specific mRNA targets in cells is of great importance in understanding gene expression and cell signaling processes. Subcellular localization of mRNA is known as a universal mechanism for cells to sequester specific mRNA for high production of required proteins. Various gene expressions in Drosophila cells are studied using quantum dots (QDs) and the fluorescence in situ hybridization (FISH) method. The excellent photostability and highly luminescent properties of QDs compared to conventional fluorophores allows reproducible obtainment of quantifiable mRNA gene expression imaging. Amine-modified oligonucleotide probes are designed and covalently attached to the carboxyl-terminated polymer-coated QDs via EDC chemistry. The resulting QD-DNA conjugates show sequence-specific hybridization with target mRNAs. Quantitative analysis of FISH on the Diptericin gene after lipopolysaccharide (LPS) treatment shows that the intensity and number of FISH signals per cell depends on the concentration of LPS and correlates well with quantitative real-time PCR results. In addition, our QD-DNA probes exhibit excellent sensitivity to detect the low-expressing Dorsal-related immunity factor gene. Importantly, multiplex FISH of Ribosomal protein 49 and Actin 5C using green and red QD-DNA conjugates allows the observation of cellular distribution of the two independent genes simultaneously. These results demonstrate that highly fluorescent and stable QD-DNA probes can be a powerful tool for direct localization and quantification of gene expression in situ.
Choi
Youngseon
Y
Nano/Bio Chemistry Group, Institut Pasteur Korea, Seongnam, South Korea.
Kim
Hwa Pyung
HP
Hong
Suk Min
SM
Ryu
Ji Young
JY
Han
Sung Jun
SJ
Song
Rita
R
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D029721:Drosophila Proteins / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry*; D045663:Quantum Dots; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; C054756:DptA protein, Drosophila; D029721:Drosophila Proteins; D011108:Polymers; D004247:DNA