Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Genome Res. 2011 Jun;21(6):925-35. doi: 10.1101/gr.116103.110. Epub 2011 May 2.
Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87-0.89) and drop-outs (4%-6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.
从单个真核细胞中进行全长转录本扩增（TTA）以进行转录组分析已得到确立，但从单个原核细胞中进行 TTA 则存在更多挑战，因为起始材料更少、缺乏 poly(A)尾巴，而且信息可能是多顺反子的。在这里，我们描述了一种使用模式生物泰国伯克霍尔德氏菌（Burkholderia thailandensis）进行单个细菌 TTA 的新方法，该细菌暴露于亚抑菌浓度的抗菌剂草甘膦中。利用 B. thailandensis 微阵列来评估 TTA 方法，与从更大规模非扩增 RNA 样本获得的数据相比，显示出低的折叠变化偏差（差异小于两倍，Pearson 相关系数 R ≈ 0.87-0.89）和缺失（2842 个可检测基因中的 4%-6%）。对微阵列数据的进一步分析表明，当泰国伯克霍尔德氏菌暴露于芳香族氨基酸生物合成抑制剂草甘膦时，会诱导（或抑制）基因，以可能恢复和平衡细胞内氨基酸池。我们分别使用 lacZ 和 gfp 报告基因融合，在多细胞和单细胞水平上验证了我们的单细胞微阵列数据。对单个细胞的 TTA 产物进行 Sanger 测序，生成了 192 个克隆，其中包括和不包括通过消除 rRNA 和 tRNA 进行富集的克隆，仅检测到无污染的泰国伯克霍尔德氏菌序列。这些数据表明，使用这种新方法可以对单个细胞的 TTA 进行 RNA-seq。
