Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

Abstract

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell-and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

“…and Leung et al 13. had respectively performed single-cell qPCR reactions in adhering droplet arrays or on-chip chambers, and showed the advantages of the microfluidic devices in cost, throughput, and precision compared with other approaches as micromanipulators35, FACS36, etc. The shortcoming of their studies lay in the need for expensive integrated equipment or sophisticated chip architecture, i.e.…”

RNA-seq based transcriptomic analysis of single bacterial cells

“…iv) generality: the key component used for RNA amplification in the BaSiC RNA-seq method, the One-Direct RNA Amplification System, was also applied in single-cell transcriptome of fungi29 and prostate cancer cells30 recently, since both poly(T) and random primers are used for the first stranded cDNA synthesis during RNA amplification, it is thus expected that with little modification, the BaSiC RNA-seq protocol can also be applied to transcriptomic profiling of eukaryotic single cells.Considering the greater challenges related to single bacterial cells (i.e., low abundance of RNA and difficult lysis), the results derived from the single-cell transcriptomics needed to be verified by other approaches. To this end, some methodologies have been recently introduced, such as pre-addition of unique molecular identifiers (UMIs)[31][32][33][34] and spike-in control RNA35 during RNA amplification or single cell FISH,31 and single cell RT-qPCR22,23 . Like most of other single-cell transcriptomics protocols established previously25,26 , BaSiC RNA-seq produces ds-cDNA, therefore cannot retain strand specificity for noncoding RNAs such as antisense RNA detection.However, the issue can be addressed by coupling the BaSiC RNA-seq analysis with single-cell RT-qPCR analysis14 , which can be employed to validate results from singlecell bacterial transcriptomic analysis.…”

Parallel RNA extraction using magnetic beads and a droplet array

“…Since PCR becomes more stochastic in the presence of fewer templates, the higher variation seen with higher C q values is an inherent characteristic of the PCR process. 31 …”