Research and Development, Illumina, Inc, San Diego, California, United States of America.
PLoS One. 2012;7(2):e30794. doi: 10.1371/journal.pone.0030794. Epub 2012 Feb 8.
We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.
METHODOLOGY/PRINCIPAL FINDINGS: The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)0.76-0.80 between individual cells and R(2)0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.
CONCLUSIONS/SIGNIFICANCE: In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.
我们开发了一种高通量扩增方法，可使用单细胞或低 RNA 输入生成稳健的基因表达谱。
方法/主要发现：该方法使用标记引物和模板转换，在合成 cDNA 的两端掺入通用 PCR 引物位点，用于全局 PCR 扩增。与全基因组基因表达微阵列平台相结合，我们通常在单个细胞之间获得 R(2)0.76-0.80 的表达相关性值，在 50pg 总 RNA 重复之间获得 R(2)0.69 的表达相关性值。来自单细胞或 50pg 总 RNA 的表达谱与更高输入（1ng 总 RNA）生成的表达谱高度相关（R(2)~0.80）。此外，该检测方法足够灵敏，可在单个细胞中检测到大约 63%的用 1ng 输入检测到的基因数量，并且在单细胞输入中检测到的大约 97%的基因也在更高输入中检测到。
结论/意义：总之，我们的方法促进了在起始材料极其有限的情况下进行全基因组基因表达谱分析，特别是在早期发育中的祖细胞研究和肿瘤干细胞生物学等领域。
