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家庭定量 mRNA 表达测量。

Quantitative mRNA expression measurement at home.

机构信息

Shanvi, San Diego, CA, USA.

Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, MC 0703, Leichtag Building 132, La Jolla, CA, 92093-0703, USA.

出版信息

Sci Rep. 2024 Jan 10;14(1):1013. doi: 10.1038/s41598-023-49651-8.

Abstract

mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.

摘要

mRNA 的测量主要依赖 RT-PCR,它需要昂贵的实验室设备和具有高等学位的人员。环介导等温扩增 (LAMP) 是一种用于检测靶 DNA 和 RNA 的多功能技术。在早期报告中,LAMP 的灵敏度低于标准 RT-PCR 检测。在这里,我们报告了使用基于荧光的 RT-LAMP 方案来测量 CDX2 表达模式,其与复杂 RT-PCR 技术的标准非常吻合(r = 0.99,p < 0.001)。该测定适用于多种样本类型,例如 cDNA、mRNA 以及直接组织样本测试,只需 25 分钟,而 RT-PCR 则需要 3 小时以上。我们已经开发了一种新的 RT-LAMP 引物设计方案,该方案可减少由于自我扩增引起的假阳性并提高定量准确性。一个带有 3D 打印盒的简单设备可实现在家庭、户外和护理点进行 mRNA 表达测量。

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