Transcript amplification from single bacterium for transcriptome analysis

Abstract

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.

Comparative Analysis of Lactobacillus plantarum WCFS1 Transcriptomes by Using DNA Microarray and Next-Generation Sequencing Technologies

“…3). In addition, the limitation of the mRNA enrichment method used (Microbexpress; Ambion), which does not target tRNA removal, resulted in the differential quantification of some tRNAs in the mRNA-enriched fraction (17). Variation in gene expression levels caused by the different growth conditions (CDM versus MRS) was observed for a total of 207 genes (FDR-adjusted P values of Ͻ0.05) from both total RNA and mRNA-enriched analyses.…”

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

“…The first single bacterial transcriptome analysis using the '29 polymerase MDA technique was reported recently by Kang et al (2011) who used Burkholderia thailandensis cells exposed to 0.01% (w/v) of glyphosate, an antibacterial agent, for single-bacterium TTA. The amplified whole transcriptome from a single B. thailandensis cell was analyzed by means of a DNA microarray.…”

“…This in situ RT-PCR approach has been successfully applied to detect gene expression in many species (Lange et al, 2000;Tolker-Nielsen et al, 1997). In addition to these traditional methods, total transcript amplification (TTA) became available for single bacterial gene expression analysis combined with microarray or messenger RNA sequencing (mRNA-Seq) (Kang et al, 2011). Single-cell based imaging methods have been improved recently and provided useful information about not only the gene expression but also the spatial information about mRNA inside a single bacterium (Taniguchi et al, 2010).…”

Exploring the oceanic microeukaryotic interactome with metaomics approaches

“…For instance, it would be possible to isolate single dinoflagellate cells infected with Amoebophrya (Coats & Park 2002 in each of the different stages of the infection to determine how the parasite attacks the host and how the host responds to the attack. Furthermore, single-cell transcriptomics could be used to analyze both eukaryotic and prokaryotic transcripts occurring in a microholobiont (Kang et al 2011), thus allowing us to explore cross-domain interactions at the intra-cellular scale.…”