Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis

Rocha, Danilo J P G; Santos, Carolina S.A.B.; Pacheco, Luis Gustavo Carvalho

doi:10.1007/s10482-015-0524-1

Cited by 142 publications

(116 citation statements)

References 36 publications

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“…As an internal validation, a gene list with housekeeping genes was composed based on genes that have been suggested by Rocha et al (2015) and Zhou et al (2011) to be used for these purposes [20,21].…”

Section: Resultsmentioning

confidence: 99%

Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

Linden

Cottyn²,

Uyttendaele

et al. 2016

Agriculture

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Numerous outbreaks of Escherichia coli O157:H7 have been linked to the consumption of leafy vegetables. However, up to the present, little has been known about E. coli O157:H7's adaptive responses to survival on actively growing (and thus responsive) plants. In this study, whole genome transcriptional profiles were generated from E. coli O157:H7 cells (isolate Sakai, stx-) one hour and two days after inoculation on the leaves of growing butterhead lettuce, and compared with an inoculum control. A total of 273 genes of E. coli O157:H7 Sakai (5.04% of the whole genome) were significantly induced or repressed by at least two-fold (p < 0.01) in at least one of the analyzed time points in comparison with the control. Several E. coli O157:H7 genes associated with oxidative stress and antimicrobial resistance were upregulated, including the iron-sulfur cluster and the multiple antibiotic resistance (mar) operon, whereas the Shiga toxin virulence genes were downregulated. Nearly 40% of the genes with significantly different expression were poorly characterized genes or genes with unknown functions. These genes are of special interest for future research as they may play an important role in the pathogens' adaptation to a lifestyle on plants. In conclusion, these findings suggest that the pathogen actively interacts with the plant environment by adapting its metabolism and responding to oxidative stress.

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Section: Resultsmentioning

confidence: 99%

Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

Linden

Cottyn²,

Uyttendaele

et al. 2016

Agriculture

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show abstract

“…To rank the candidate reference genes from the most to least stably expressed and to select the most useful for determining the expression of pathogenicity-related genes ( amsB and hrpN ), several different mathematical algorithms were adopted 12, 40, 43, 46, 47 . The comprehensive ranging obtained based on the results of all the algorithms used showed that proC , followed by recA and ffh , were the most stably expressed set of reference genes.…”

Section: Discussionmentioning

confidence: 99%

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

Kałużna

Kuras

Puławska

2017

Sci Rep

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To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.

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“…The cDNA was synthesized from 1 μg of purified RNA with Bio-Rad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, United States), and quantitative amplification condition using Bio-Rad iTaq Universal SYBR Green Supermix and Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc.). Standard curves for each primer were used to determine the relative number of cDNA molecules, and relative expression was calculated by normalizing to the gyrA gene transcripts, which is a validated reference gene for normalization of qRT-PCR (Rocha et al, 2015; Zeng and Burne, 2016). The minimum information for publication of qRT-PCR experiments (MIQE) guidelines were followed for quality control of the data generated and for data analysis (Bustin et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

Kim

Zhou

et al. 2017

Front. Microbiol.

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Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH) genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins) and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial–fungal interactions may enhance the severity of dental caries.

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Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis

Cited by 142 publications

References 36 publications

Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

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