Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe
Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe
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使用双筒碳探针从组织模型中分离和定量信使 RNA
DOI：
10.1007/s00216-013-7430-z
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
梨本裕司・高橋康史・高野亮介・宮下紘介・山田淑代・伊野浩介・珠玖仁・末永智一
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文献类型：
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作者：
Shibayama;M.;梨本裕司・高橋康史・高野亮介・宮下紘介・山田淑代・伊野浩介・珠玖仁・末永智一
在这项研究中，我们引入了双筒碳探针（DBCP）--一种简单、经济实惠的微环电极--它能够独立于细胞定位来收集和分析单细胞。通过在DBCP中的两个电极之间利用电脉冲来穿刺靶细胞，并且通过使用DBCP手动抽吸来收集细胞裂解物。使用实时PCR定量评价收集的裂解物中的mRNA。将归一化为GAPDH的单细胞相对基因表达的直方图拟合为理论对数正态分布。在组织培养模型中，我们着重于血管生成，以证明多基因表达分析是可行的。最后，我们将DBCP应用于胚胎干（ES）细胞衍生的心肌细胞，以证实探针收集细胞的能力，即使是从大体积样品，如球体。该方法在单细胞水平上实现了对mRNA的高灵敏度，并且适用于独立于细胞定位的各种生物样品的分析。
In this study, we introduce the double-barrel carbon probe (DBCP)—a simple, affordable microring electrode—which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.