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Sherif M Shawky,1–3,* Ahmed M Awad,1,4,* Arwa A Abugable,1,2,* Sherif F El-Khamisy1,2 1Center for Genomics, Helmy Institute for Medical Sciences, Zewail City of Science and Technology, Giza, Egypt; 2Krebs Institute, Department ofMolecular Biology and Biotechnology, Firth Court, University of Sheffield, Sheffield, UK; 3Biochemistry Department, Faculty of Pharmacy, Misr University for Science and Technology, Giza, Egypt; 4Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt *These authors contributed equally tothis work Purpose: The objective of this study is to develop a facile tool for the absolute detection and quantification of nucleic acid transcripts, using a gold nanoparticle-based optical biosensor. Topoisomerase 1 (TOP1) and tyrosyl DNA phosphodiesterase 2 (TDP2) were among the nucleic acid transcripts of choice due to their role as genomic instability biomarkers and their implication in various cancers and neurologic disorders. This opens the door to develop a simple tool that can be used for diagnosing and monitoring treatment response for such diseases, overcoming the requirements for high cost, time, and complexity of the existing technologies for the absolute quantification of transcripts of interest. Materials and methods: The TOP1 and TDP2 mRNA transcripts were first captured specifically using magnetic nanoparticles that were functionalized with TOP1- and TDP2-specific probes, respectively. The captured mRNA was then directly detected and quantified using the gold aggregating gold (GAG) assay, without the need for amplification as in existing technologies used for the quantification of transcripts. Results: A linear correlation exists between the GAG assay and the qPCR for the quantification of the TOP1 and TDP2 mRNA transcripts (101–104 copies). The detection limit of the GAG assay in mRNA quantification was up to 10 copies per reaction. Wild-type and TDP2-deficient cell lines confirmed the assay specificity and reproducibility in distinguishing between different transcripts. Conclusion: The GAG assay can be utilized as an inexpensive, rapid, simple, and sensitive tool for the absolute quantification of RNA for different applications, instead of the laborious, expensive, and sophisticated real-time PCR. Keywords: gold aggregating gold, TOP1, TDP2, magnetic nanoparticles, RNA extraction, genomic instability, neurodegenerative diseases, tumor markers |
