Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent
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 reacti...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/1/347 |
| _version_ | 1797358696503705600 |
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| author | Oleksij Redcenko Magda Tumova Petr Draber |
| author_facet | Oleksij Redcenko Magda Tumova Petr Draber |
| author_sort | Oleksij Redcenko |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T15:05:45Z |
| format | Article |
| id | doaj.art-e1c9c84f492648e9923726658d0eb789 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T15:05:45Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-e1c9c84f492648e9923726658d0eb7892024-01-10T14:59:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0125134710.3390/ijms25010347Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking AgentOleksij Redcenko0Magda Tumova1Petr Draber2Laboratory of Signal Transduction, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech RepublicLaboratory of Signal Transduction, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech RepublicLaboratory of Signal Transduction, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech RepublicDue 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.https://www.mdpi.com/1422-0067/25/1/347DNA aptamerspolymerase chain reactionpolypropylene wellsimmunoglobulinsblocking agentsmethylcellulose |
| spellingShingle | Oleksij Redcenko Magda Tumova Petr Draber Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent International Journal of Molecular Sciences DNA aptamers polymerase chain reaction polypropylene wells immunoglobulins blocking agents methylcellulose |
| title | Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent |
| title_full | Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent |
| title_fullStr | Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent |
| title_full_unstemmed | Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent |
| title_short | Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent |
| title_sort | simplified pcr based quantification of proteins with dna aptamers and methylcellulose as a blocking agent |
| topic | DNA aptamers polymerase chain reaction polypropylene wells immunoglobulins blocking agents methylcellulose |
| url | https://www.mdpi.com/1422-0067/25/1/347 |
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