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Absolute quantification of cell-bound DNA aptamers during SELEX
Meltem Avci-Adali1, Nadja Wilhelm, Nadja Perle
1Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen , Tuebingen, Germany.
Nucleic Acid Therapeutics
|February 15, 2013
Summary
A new quantitative real-time PCR (qPCR) method accurately measures aptamers bound to cells during cell-SELEX. This technique provides absolute quantification, improving aptamer selection and preventing contamination in diagnostic and therapeutic applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Nucleic Acid Chemistry
Background:
- Aptamers are nucleic acid ligands with applications in diagnostics, imaging, regenerative medicine, and drug targeting.
- Cell-SELEX is a method for selecting aptamers that bind to specific cells.
- Current monitoring of cell-SELEX uses flow cytometry, which offers only relative quantification and requires labeled oligonucleotides.
Purpose of the Study:
- To develop and validate a novel quantitative real-time PCR (qPCR) method for absolute determination of cell-bound aptamers during cell-SELEX.
- To establish a more accurate and sensitive monitoring tool for aptamer selection compared to existing methods.
Main Methods:
- Development of a SYBR Green I-based real-time PCR assay.
- Creation of an aptamer standard curve for absolute quantification.
- Lysates of cells with bound aptamers were analyzed to determine the absolute aptamer quantity.
Main Results:
- The developed qPCR method provides absolute quantification of cell-bound aptamers.
- The method demonstrates high sensitivity, with a lower detection limit of 20 fg.
- The assay allows for accurate monitoring of aptamer enrichment during cell-SELEX.
Conclusions:
- The validated qPCR method serves as an effective tool for monitoring aptamer enrichment during cell-SELEX.
- Absolute quantification using qPCR enhances the precision of aptamer selection.
- This method can help prevent contamination of amplified aptamer pools with by-products.
