Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method
Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method
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DOI：
10.1039/c2an15866c
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发表时间：
2012-01-01
期刊：
影响因子：
4.2
通讯作者：
Kubo, Izumi
中科院分区：
文献类型：
--
作者：
Furutani, Shunsuke;Nagai, Hidenori;Kubo, Izumi
为了能够检测单个细胞中基因的表达，在一个设备中分离和裂解单个细胞并进行逆转录聚合酶链反应（RT-PCR）的能力是很重要的。通常，在同一反应室中进行细胞裂解和RT-PCR时，需要在细胞裂解后添加RT-PCR试剂。在这项研究中，我们提出了一个原始的公式，细胞裂解和RT-PCR在同一个反应室不添加试剂，只有一个加热过程，我们称之为热细胞直接RT-PCR。热细胞直接RT-PCR通过使用Tth DNA聚合酶实现，Tth DNA聚合酶是一种耐热聚合酶，在锰离子存在下具有高逆转录活性。使用双染料荧光探针检测荧光，直接检测RT-PCR产物。我们尝试用微流体装置检测分离Jurkat细胞中甘油醛-3-磷酸脱氢酶（GAPDH）基因的mRNA，我们已经开发了用于单细胞分离的微流体装置。细胞分离后，在设备上进行连续的热细胞直接RT-PCR，检测单个细胞的RT-PCR产物的荧光信号，并与不含细胞的室进行分化。含有细胞的室数与含有RT-PCR产物的室数在10 ~ 400个细胞μ L-1之间呈高度正线性关系（r = 0.9933）。因此，利用新型的热细胞直接RT-PCR方法检测分离细胞中表达的基因成为可能。
In order to be able to detect the expression of a gene in individual cells, the ability to isolate and lyse a single cell and to perform reverse transcription polymerase chain reaction (RT-PCR) in one device is important. As is common, when performing cell lysis and RT-PCR in the same reaction chamber, it is necessary to add the reagent for RT-PCR after cell lysis. In this study, we propose an original formula for cell lysis and RT-PCR in the same reaction chamber without the addition of reagent by only a heat process, which we termed hot cell-direct RT-PCR. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase, which is a thermostable polymerase and has high reverse transcription activity in the presence of manganese ions. Direct detection of RT-PCR products was performed by detecting fluorescence with the use of a double-dye fluorescent probe. We attempted to detect the mRNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in isolated Jurkat cells on a microfluidic device, which we had already developed for single cell isolation. After cell isolation and successive hot cell-direct RT-PCR on the device, fluorescent signals from RT-PCR products for a single cell were detected and differentiated from the chamber containing no cells. A highly positive linear relationship (r = 0.9933) was observed between the number of chambers containing cell(s) and those containing RT-PCR products from 10 to 400 cells mu L-1. Thus it was possible to use the novel hot cell-direct RT-PCR method to detect the expressed gene in isolated cells.