RT-qPCR based quantitative analysis of gene expression in single bacterial cells

Gao, Weimin; Zhang, Weiwen; Meldrum, Deirdre R.

doi:10.1016/j.mimet.2011.03.008

Cited by 61 publications

(59 citation statements)

References 47 publications

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“…Primers for RT-qPCR were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast /index.cgi?LINK_LOCϭBlastHome). To differentiate PCR products from primer dimers, we selected primers which will generate amplicons with sizes around 170 to 220 bp (37). qPCR was performed using Express SYBR GreenER qPCR SuperMix kits (Invitrogen, Carlsbad, CA) on an ABI StepOne real-time PCR system for bulk-cell analysis and an ABI 7900HT real-time PCR system for single-cell analysis (Applied Biosystems, Foster, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Nevertheless, due to their key roles in the ecology and biogeochemistry of the oceans, it is important to further understand the mechanism that diatoms use to deal with various environmental stresses. In this study, Thalassiosira pseudonana (35), a typical centric diatom, was applied as a model system to measure the stress response of microorganisms to their environment at the singlecell level using single-cell RT-qPCR, based on our previous efforts (36,37). In contrast to previously published single-cell analyses on mammalian or prokaryotic cells, working with diatoms has its own particular challenges due to their small size (ϳ5-m diameter) and protective frustules.…”

mentioning

confidence: 99%

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

Shi

Gao

Chao

et al. 2013

Appl Environ Microbiol

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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.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Shi

Gao

Chao

et al. 2013

Appl Environ Microbiol

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“…Protein interactions, Spatiotemporal dynamics (Suhling et al, 2005) (Hutchison and Venter, 2006;Lasken and McLean, 2014;Saliba et al, 2014) PCR qPCR, RT-qPCR, In situ PCR Gene expression, Single cell identification and characterization (Gao et al, 2011;Hodson et al, 1995;Shi et al, 2011) Fluorescence in situ hybridizationImmunofluorescence -Avi et al, 2014;Davey and Kell, 1996;Koch et al, 2014;Shapiro, 2000) …”

Section: Fluorescence Lifetime Imaging Microscopy (Flim)mentioning

confidence: 99%

Individual cell heterogeneity in Predictive Food Microbiology: Challenges in predicting a “noisy” world

Koutsoumanis

Aspridou

2017

International Journal of Food Microbiology

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“…Thus, this sequential isolation can help to reduce the background of the non-ice nucleation active microbial community in the sample and provides a first step towards an identification of most active IN. At that stage, either selective cultivation-based methods may allow the recovery of the biological agent responsible for the nucleation, or molecular approaches such as amplification of key genes may be applied, since extraction and amplification of DNA even from single cells seems to become an increasingly feasible method (Gao et al, 2011).…”

Section: Progressive Isolation Of Ice Nucleators From a Samplementioning

confidence: 99%

Freezing nucleation apparatus puts new slant on study of biological ice nucleators in precipitation

et al. 2014

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Abstract. For decades, drop-freezing instruments have contributed to a better understanding of biological ice nucleation and its likely implications for cloud and precipitation development. Yet, current instruments have limitations. Drops analysed on a cold stage are subject to evaporation and potential contamination. The use of closed tubes provides a partial solution to these problems, but freezing events are still difficult to be clearly detected. Here, we present a new apparatus where freezing in closed tubes is detected automatically by a change in light transmission upon ice development, caused by the formation of air bubbles and crystal facets that scatter light. Risks of contamination and introduction of biases linked to detecting the freezing temperature of a sample are then minimized. To illustrate the performance of the new apparatus we show initial results of two assays with snow samples. In one, we repeatedly analysed the sample (208 tubes) over the course of a month with storage at +4 • C, during which evidence for biological ice nucleation activity emerged through an increase in the number of ice nucleators active around −4 • C. In the second assay, we indicate the possibility of increasingly isolating a single ice nucleator from a precipitation sample, potentially determining the nature of a particle responsible for a nucleation activity measured directly in the sample. These two seminal approaches highlight the relevance of this handy apparatus for providing new points of view in biological ice nucleation research.

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RT-qPCR based quantitative analysis of gene expression in single bacterial cells

Cited by 61 publications

References 47 publications

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

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

Individual cell heterogeneity in Predictive Food Microbiology: Challenges in predicting a “noisy” world

Freezing nucleation apparatus puts new slant on study of biological ice nucleators in precipitation

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