Korfhage Christian, Fricke Evelyn, Meier Andreas
Qiagen, Hilden, Germany.
Curr Protoc Mol Biol. 2015 Jul 1;111:7.19.1-7.19.18. doi: 10.1002/0471142727.mb0719s111.
Genomic DNA determines how and when the transcriptome is changed by a trigger or environmental change and how cellular metabolism is influenced. Comparative genome and transcriptome analysis of the same cell sample links a defined genome with all changes in the bases, structure, or numbers of the transcriptome. However, comparative genome and transcriptome analysis using next-generation sequencing (NGS) or real-time PCR is often limited by the small amount of sample available. In mammals, the amount of DNA and RNA in a single cell is ∼10 picograms, but deep analysis of the genome and transcriptome currently requires several hundred nanograms of nucleic acids for library preparation for NGS sequencing. Consequently, accurate whole-genome amplification (WGA) and whole-transcriptome amplification (WTA) is required for such quantitative analysis. This unit describes how the genome and the transcriptome of a tiny number of cells can be amplified in a highly parallel and comparable process. Protocols for quality control of amplified DNA and application of amplified DNA for NGS are included.
基因组DNA决定了转录组如何以及何时因触发因素或环境变化而改变,以及细胞代谢如何受到影响。对同一细胞样本进行比较基因组和转录组分析,可将特定的基因组与转录组中碱基、结构或数量的所有变化联系起来。然而,使用下一代测序(NGS)或实时PCR进行比较基因组和转录组分析,往往受到可用样本量少的限制。在哺乳动物中,单个细胞中的DNA和RNA量约为10皮克,但目前对基因组和转录组的深入分析需要数百纳克核酸用于NGS测序的文库制备。因此,进行此类定量分析需要准确的全基因组扩增(WGA)和全转录组扩增(WTA)。本单元描述了如何在高度平行且可比的过程中扩增少量细胞的基因组和转录组。其中还包括扩增DNA的质量控制协议以及扩增DNA在NGS中的应用。