Simple and efficient transcriptome profiling from a single embryo

Measuring the global gene expression pattern in a single cell has been technically challenging, but is potentially very useful for understanding variation in biological processes between cells and for studying gene regulation during early embryo development with single cell resolution. To advance these applications, multiple systems for single-cell RNA extraction in addition to crude cell lysis followed by RNA profiling were examined and used for genomic data analysis. Our results indicate that some of these methods are suitable for analyzing even a portion of the total RNA from a single oocyte or embryo, with low variance among technical replicates across a wide dynamic range of expression. These methods will not only be helpful for genomic and epigenetic research to identify regulatory mechanisms of early development and molecular signatures of embryo classes, but can also provide great potential for clinical analysis of small biopsy samples or pre-implantation genetic disease screening. RNA from single mouse oocytes was purified using Qiagen Rneasy Mini or Arcturus PicoPure kits. Crude lysates of single oocytes were also prepared in NuGEN direct lysis buffer. Three replicate oocytes for each of the three sample preparation methods were used. Total RNA was amplified by NuGEN Ovation One-Direct ribo-SPIA, cDNA products were biotinylated, and labeled targets were hybridized to Affymetrix Mouse Gene 1.0ST Arrays.对单个细胞开展全局基因表达谱检测曾在技术层面极具挑战性，但该技术对于解析细胞间生物学过程的差异，以及以单细胞分辨率研究早期胚胎发育过程中的基因调控机制，均具备极高的应用价值。为推动上述应用的发展，本研究对除粗制细胞裂解后进行RNA谱分析之外的多种单细胞RNA提取体系进行了测试，并将其应用于基因组数据分析。研究结果表明，部分方法即便仅使用单个卵母细胞或胚胎的总RNA的部分量，也可实现有效分析，且在宽广的表达动态范围内，技术重复间的变异度极低。此类方法不仅可助力基因组学与表观遗传学研究，以识别早期发育的调控机制及胚胎类别的分子特征，还可为小型活检样本的临床分析以及植入前遗传病筛查提供巨大应用潜力。本研究使用Qiagen Rneasy Mini试剂盒与Arcturus PicoPure试剂盒对单个小鼠卵母细胞的RNA进行纯化。同时，本研究还通过NuGEN直接裂解缓冲液制备了单个卵母细胞的粗裂解液。针对三种样本制备方法，每种方法均使用三个重复卵母细胞样本。采用NuGEN Ovation One-Direct ribo-SPIA技术对总RNA进行扩增，将cDNA产物进行生物素标记，并将标记后的靶标与Affymetrix Mouse Gene 1.0ST基因芯片进行杂交。