Lao Kai Q, Tang Fuchou, Barbacioru Catalin, Wang Yangzhou, Nordman Ellen, Lee Clarence, Xu Nanlan, Wang Xiaohui, Tuch Brain, Bodeau John, Siddiqui Asim, Surani M Azim
Applied Biosystems, a division of Life Technologies Corporation, Genetic Analysis Business Unit, Foster City, California 94404, USA.
J Biomol Tech. 2009 Dec;20(5):266-71.
We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.
我们通过将改良的单细胞全转录组扩增方法与新一代测序技术SOLiD系统相结合，开发了一种基于测序的单细胞分辨率基因表达谱分析方法。使用该分析方法，我们发现四细胞期胚胎中的卵裂球具有相似的基因表达，这与它们具有相似的发育潜能这一事实相符。我们证明，与cDNA微阵列技术相比，我们的单细胞cDNA SOLiD测序分析方法能够检测出数千个更多基因的表达。此外，对于微阵列和SOLiD测序检测到的基因，我们的分析方法在很大比例的基因中检测到了新的转录变体，这在单细胞分辨率上明确证实了转录组复杂性高于传统预期。最后，通过将我们的分析方法应用于Dicer基因敲除（KO）和Ago2基因敲除的卵母细胞，我们发现与野生型对照相比，Dicer/Ago2基因敲除的成熟卵母细胞中有大量转座子异常上调。
