Rapid quantification of microRNAs in plasma using a fast real-time PCR system
The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers for a range of conditions. One rate-limiting factor in miRNA detection is the time taken to perform quantitative real-time PCR (qPCR) ampli...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2015-05-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114287 |
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| _version_ | 1850152474388725760 |
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| author | William John Andrews Eoin Daniel Brown Margaret Dellett Ruth Esther Hogg David Arthur Simpson |
| author_facet | William John Andrews Eoin Daniel Brown Margaret Dellett Ruth Esther Hogg David Arthur Simpson |
| author_sort | William John Andrews |
| collection | DOAJ |
| description | The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers for a range of conditions. One rate-limiting factor in miRNA detection is the time taken to perform quantitative real-time PCR (qPCR) amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress thermal cycler (BJS Biotechnologies), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10°C/s, and a conventional Peltier-controlled LightCycler 480 system (Roche) ramping at 4.8°C/s. The quantification cycle (Cq) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, P = 2.4 × 10-25) for the xxpress (20.01 ± 0.47 sd) than for the LightCycler (19.87 ± 0.04 sd). RNA was extracted from human plasma, reverse transcribed, and a panel of miRNAs was amplified and detected using SYBR Green. The sensitivities of the two systems were broadly comparable–both detected a panel of miRNAs reliably, and both indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of-care diagnostics based upon detection of circulating miRNAs a step closer to reality. |
| format | Article |
| id | doaj-art-98809270800b49ccb199fc4cdb5fbda4 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-05-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-98809270800b49ccb199fc4cdb5fbda42025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-05-0158524425210.2144/000114287Rapid quantification of microRNAs in plasma using a fast real-time PCR systemWilliam John Andrews0Eoin Daniel Brown1Margaret Dellett2Ruth Esther Hogg3David Arthur Simpson41Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom1Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom1Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom1Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom1Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United KingdomThe ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers for a range of conditions. One rate-limiting factor in miRNA detection is the time taken to perform quantitative real-time PCR (qPCR) amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress thermal cycler (BJS Biotechnologies), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10°C/s, and a conventional Peltier-controlled LightCycler 480 system (Roche) ramping at 4.8°C/s. The quantification cycle (Cq) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, P = 2.4 × 10-25) for the xxpress (20.01 ± 0.47 sd) than for the LightCycler (19.87 ± 0.04 sd). RNA was extracted from human plasma, reverse transcribed, and a panel of miRNAs was amplified and detected using SYBR Green. The sensitivities of the two systems were broadly comparable–both detected a panel of miRNAs reliably, and both indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of-care diagnostics based upon detection of circulating miRNAs a step closer to reality.https://www.future-science.com/doi/10.2144/000114287PCRmicroRNART-PCRbiomarker |
| spellingShingle | William John Andrews Eoin Daniel Brown Margaret Dellett Ruth Esther Hogg David Arthur Simpson Rapid quantification of microRNAs in plasma using a fast real-time PCR system BioTechniques PCR microRNA RT-PCR biomarker |
| title | Rapid quantification of microRNAs in plasma using a fast real-time PCR system |
| title_full | Rapid quantification of microRNAs in plasma using a fast real-time PCR system |
| title_fullStr | Rapid quantification of microRNAs in plasma using a fast real-time PCR system |
| title_full_unstemmed | Rapid quantification of microRNAs in plasma using a fast real-time PCR system |
| title_short | Rapid quantification of microRNAs in plasma using a fast real-time PCR system |
| title_sort | rapid quantification of micrornas in plasma using a fast real time pcr system |
| topic | PCR microRNA RT-PCR biomarker |
| url | https://www.future-science.com/doi/10.2144/000114287 |
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