Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions
The fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we a...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-05-01
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| Series: | Micromachines |
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| Online Access: | http://www.mdpi.com/2072-666X/9/6/272 |
| _version_ | 1818923247915237376 |
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| author | Yuri Ishigaki Kae Sato |
| author_facet | Yuri Ishigaki Kae Sato |
| author_sort | Yuri Ishigaki |
| collection | DOAJ |
| description | The fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we aimed to improve the efficiency of padlock/RCA by determining the effects of microchannel shape and ultrasonic solution mixing. Using a circular-shaped microchamber and ultrasonic mixing, the efficiency of microfluidic padlock/RCA was improved, and the consumption of the expensive probe solution was reduced from 10 µL to approximately 3.5 µL. Moreover, the fluorescent probe hybridization time was reduced to 5 min, which is four times faster than that of the standard protocol. We used this method to successfully detect mitochondrial DNA and transcripts of β-actin and K-ras proto-oncogene codon 12 in cells. Our method offers improvements over current padlock/RCA methods and will be helpful in optimizing other microfluidics-based FISH-related analyses. |
| first_indexed | 2024-12-20T02:06:25Z |
| format | Article |
| id | doaj.art-1fd4c5196bd546dbb7c8a03eae7137c6 |
| institution | Directory Open Access Journal |
| issn | 2072-666X |
| language | English |
| last_indexed | 2024-12-20T02:06:25Z |
| publishDate | 2018-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Micromachines |
| spelling | doaj.art-1fd4c5196bd546dbb7c8a03eae7137c62022-12-21T19:57:10ZengMDPI AGMicromachines2072-666X2018-05-019627210.3390/mi9060272mi9060272Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) ReactionsYuri Ishigaki0Kae Sato1Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Bunkyo, Tokyo 112-8681, JapanDepartment of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, Bunkyo, Tokyo 112-8681, JapanThe fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we aimed to improve the efficiency of padlock/RCA by determining the effects of microchannel shape and ultrasonic solution mixing. Using a circular-shaped microchamber and ultrasonic mixing, the efficiency of microfluidic padlock/RCA was improved, and the consumption of the expensive probe solution was reduced from 10 µL to approximately 3.5 µL. Moreover, the fluorescent probe hybridization time was reduced to 5 min, which is four times faster than that of the standard protocol. We used this method to successfully detect mitochondrial DNA and transcripts of β-actin and K-ras proto-oncogene codon 12 in cells. Our method offers improvements over current padlock/RCA methods and will be helpful in optimizing other microfluidics-based FISH-related analyses.http://www.mdpi.com/2072-666X/9/6/272microfluidicspadlock proberolling circle amplificationfluorescent hybridization |
| spellingShingle | Yuri Ishigaki Kae Sato Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions Micromachines microfluidics padlock probe rolling circle amplification fluorescent hybridization |
| title | Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions |
| title_full | Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions |
| title_fullStr | Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions |
| title_full_unstemmed | Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions |
| title_short | Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions |
| title_sort | effects of microchannel shape and ultrasonic mixing on microfluidic padlock probe rolling circle amplification rca reactions |
| topic | microfluidics padlock probe rolling circle amplification fluorescent hybridization |
| url | http://www.mdpi.com/2072-666X/9/6/272 |
| work_keys_str_mv | AT yuriishigaki effectsofmicrochannelshapeandultrasonicmixingonmicrofluidicpadlockproberollingcircleamplificationrcareactions AT kaesato effectsofmicrochannelshapeandultrasonicmixingonmicrofluidicpadlockproberollingcircleamplificationrcareactions |