Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent

0581638 - ÚMG 2025 RIV CH eng J - Článek v odborném periodiku
Redčenko, Oleksij - Tůmová, Magda - Dráber, Petr
Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent.
International Journal of Molecular Sciences. Roč. 25, č. 1 (2024), č. článku 347. ISSN 1661-6596
Grant CEP: GA TA ČR(CZ) TG01010066; GA TA ČR TP01010060; GA TA ČR TA01010436; GA ČR(CZ) GA23-07736S; GA MPO FV20479; GA MŠMT LM2023052
Institucionální podpora: RVO:68378050
Klíčová slova: blocking agents * DNA aptamers * immunoglobulins * methylcellulose * polymerase chain reaction * polypropylene wells
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 5.6, rok: 2022
Způsob publikování: Open access
https://mdpi.com/1422-0067/25/1/347

Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.
Trvalý link: https://hdl.handle.net/11104/0349748