Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

Zaitoun, Ismail; Erickson, Christopher S.; Schell, Kathleen; Epstein, Miles L.

doi:10.1186/1756-0500-3-328

Cited by 15 publications

(13 citation statements)

References 27 publications

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“…2.5a). Furthermore, in contrast to a recently published report (Zaitoun et al, 2010), RNAlater did not seem to interfere with GFP fluorescence (Fig. 2.5c).…”

Section: Transcriptome Fixationcontrasting

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…2.5a). Furthermore, in contrast to a recently published report (Zaitoun et al, 2010), RNAlater did not seem to interfere with GFP fluorescence (Fig. 2.5c).…”

Section: Transcriptome Fixationcontrasting

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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“…At 69 h detached cells (DC) were collected from the culture medium supernatant. To preserve RNA integrity during FACS sorting of infected cells, cells were treated with RNAlater (27,28). In addition, sporozoite samples were generated by processing infected salivary glands of mosquitoes at day 20 post feeding.…”

Section: Resultsmentioning

confidence: 99%

“…The pellet was loosened by flicking and the cells were resuspended with 250ul of RNAlater and stored at 4°C till all time points were harvested. It has been reported that RFP in contrast to GFP is preserved in RNAlater treated cells (28).…”

Section: Methodsmentioning

confidence: 99%

Transcriptome analysis ofPlasmodium bergheiduring exo-erythrocytic development

Caldelari

Dogga

Schmid³

et al. 2019

Preprint

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SummaryThe complex life cycle of malaria parasites requires well-orchestrated stage specific gene expression. In the vertebrate host the parasites grow and multiply by schizogony in two different environments: within erythrocytes and within hepatocytes. Whereas erythrocytic parasites are rather well-studied in this respect, relatively little is known about the exo-erythrocytic stages. In an attempt to fill this gap, we performed genome wide RNA-seq analyses of various exo-erythrocytic stages of Plasmodium berghei including sporozoites, samples from a time-course of liver stage development and detached cells, which contain infectious merozoites and represent the final step in exo-erythrocytic development. The analysis represents the completion of the transcriptome of the entire life cycle of P. berghei parasites with temporal detailed analysis of the liver stage allowing segmentation of the transcriptome across the progression of the life cycle. We have used these RNA-seq data from different developmental stages to cluster genes with similar expression profiles, in order to infer their functions. A comparison with published data of other parasite stages confirmed stage-specific gene expression and revealed numerous genes that are expressed differentially in blood and exo-erythrocytic stages. One of the most exo-erythrocytic stage-specific genes was PBANKA_1003900, which has previously been annotated as a “gametocyte specific protein”. The promoter of this gene drove high GFP expression in exo-erythrocytic stages, confirming its expression profile seen by RNA-seq. The comparative analysis of the genome wide mRNA expression profiles of erythrocytic and different exo-erythrocytic stages improves our understanding of gene regulation of Plasmodium parasites and can be used to model exo-erythrocytic stage metabolic networks and identify differences in metabolic processes during schizogony in erythrocytes and hepatocytes.

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“…Samples were then spun down at 37 °C, 300g for 10 minutes and resuspended in 1 ml PBS + 2% FCS and placed on ice until FACS. RNAlater was used shortly after sorting, as this reagent leads to decreased GFP expression 49 . To control for gene expression changes that may be induced by FACS, only sorted samples were used in comparative analyses.…”

Section: Methodsmentioning

confidence: 99%

Coordinated host-pathogen transcriptional dynamics revealed using sorted subpopulations and single,Candida albicansinfected macrophages

Muñoz

Delorey

Ford

et al. 2018

Preprint

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13The control of fungal infections depends on interactions with innate immune cells including 14 macrophages, which are among the first host cell types to respond to pathogens such as Candida 15albicans. This fungus is a member of the healthy human microbiome, although also a devastating 16 pathogen in immunocompromised individuals. Consistent with recent findings from studies of other 17 pathogens, we observed that within a population of interacting macrophages and C. albicans, there are 18 distinct host-pathogen subpopulations reflecting cell specific trajectories and infection outcomes. Little 19 is known about the molecular mechanisms that control these different fates. To address this, we 20 developed an experimental system to isolate the major host-fungal pathogen subpopulations observed 21 during ex vivo infection using fluorescent markers. We separated subpopulations of macrophages 22 infected with live C. albicans from uninfected cells and assessed the variability of gene expression in 23 both host and fungal pathogen for each subpopulation across time using RNA-Seq. In infected cells, we 24 observed a coordinated, time-dependent shift in gene expression for both host and fungus. The early 25 response in macrophages was established upon exposure to C. albicans prior to engulfment and 26 involved up-regulation of pathways and regulatory genes required for cell migration, pathogen 27 recognition, activation of engulfment, and phagocytosis; this pro-inflammatory response declined during 28 later time points in parallel with expression changes in C. albicans. After phagocytosis, the initial 29 response of C. albicans was to up-regulate genes related to survival in the nutrient-limited and stressful 30 environment within macrophages; at later time points, gene expression shifted to initiate hyphal growth 31 and escape. To further probe the heterogeneity seen observed in host-pathogen interactions, we 32 performed RNA-Seq of single macrophages infected with C. albicans. We observed that some genes 33 show higher levels of heterogeneity in both host and fungal pathogen cells that we could not detect in 34 subpopulation samples; we observed that the time shift in expression is asynchronous and that 35 expression changes in both the host and pathogen are tightly coupled. This work highlights how 36 . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . http://dx.doi.org/10.1101/350322 doi: bioRxiv preprint first posted online Jun. 19, 2018; 3To overcome these challenges, we developed an experimental system to isolate subpopulations of 70 distinct infection outcomes and examined host and pathogen gene expression in parallel, in sorted 71 subpopulations a...

show abstract

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

Cited by 15 publications

References 27 publications

Dual RNA-seq of pathogen and host

Dual RNA-seq of pathogen and host

Transcriptome analysis ofPlasmodium bergheiduring exo-erythrocytic development

Coordinated host-pathogen transcriptional dynamics revealed using sorted subpopulations and single,Candida albicansinfected macrophages

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