Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2021-05-28 , DOI: 10.1007/s00216-021-03415-3 Ying Zhou 1 , Erteng Jia 1 , Yi Qiao 1 , Huajuan Shi 1 , Zhiyu Liu 1 , Min Pan 2 , Xiangwei Zhao 1 , Yunfei Bai 1 , Qinyu Ge 1
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Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30 μm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.
Graphical abstract
中文翻译：
基于琼脂糖凝胶的带限制效应的低偏置多重置换扩增
多重置换扩增（MDA）是一种流行的单细胞全基因组扩增（WGA）技术，可以大大提高单细胞基因组的扩增效率。但是，存在一个无法彻底解决的固有问题，即放大偏差。我们在这里提出了一种基于低熔点琼脂糖凝胶的改进 MDA 方法，称为 gelMDA。首先，琼脂糖凝胶和溶液用扫描电镜和荧光试剂进行表征。然后，我们在RNA-seq的文库制备中使用gelMDA进行cDNA扩增，并使用常规MDA作为比较。通过荧光曲线、产物产量和测序结果评估gelMDA的灵敏度、效率和扩增偏差。最后，gelMDA 用于单细胞转录组测序。结果表明，gelMDA的灵敏度和产率明显高于常规MDA。从 gelMDA 测序结果中获得了较低的变异系数 (CV) 和较高的重现性。从组织切片中扩增出直径为 30 μm 的区域并成功测序。总之，gelMDA 在本研究中获得了更高的扩增效率和更低的扩增偏差。它表明单细胞 RNA 扩增和测序的巨大潜力。在本研究中，gelMDA 获得了更高的扩增效率和更低的扩增偏差。它表明单细胞 RNA 扩增和测序的巨大潜力。在本研究中，gelMDA 获得了更高的扩增效率和更低的扩增偏差。它表明单细胞 RNA 扩增和测序的巨大潜力。
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