Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Chong, Gabriella; Kuo, Fu‐Wen; Tsai, Shu-Yao; Lin, Chiahsin

doi:10.1038/srep39396

Cited by 16 publications

(10 citation statements)

References 55 publications

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“…Most of these genes function in catalytic activities and binding (Figure 1), which are essential for metabolism and growth in the organism. To date, only a few stably expressed housekeeping genes have been identified in Symbiodiniaceae, as candidates of reference genes with which to normalize gene expression in molecular studies [69,70]. The 221 stably and highly expressed core genes identified in this study add more candidates for reference genes in future gene expression studies on F. kawagutii .…”

Section: Discussionmentioning

confidence: 96%

Transcriptomic Responses to Thermal Stress and Varied Phosphorus Conditions in Fugacium kawagutii

Lin

Zhang

2019

Microorganisms

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Coral reef-associated Symbiodiniaceae live in tropical and oligotrophic environments and are prone to heat and nutrient stress. How their metabolic pathways respond to pulses of warming and phosphorus (P) depletion is underexplored. Here, we conducted RNA-seq analysis to investigate transcriptomic responses to thermal stress, phosphate deprivation, and organic phosphorus (OP) replacement in Fugacium kawagutii. Using dual-algorithm (edgeR and NOIseq) to remedy the problem of no replicates, we conservatively found 357 differentially expressed genes (DEGs) under heat stress, potentially regulating cell wall modulation and the transport of iron, oxygen, and major nutrients. About 396 DEGs were detected under P deprivation and 671 under OP utilization, both mostly up-regulated and potentially involved in photosystem and defensome, despite different KEGG pathway enrichments. Additionally, we identified 221 genes that showed relatively stable expression levels across all conditions (likely core genes), mostly catalytic and binding proteins. This study reveals a wide range of, and in many cases previously unrecognized, molecular mechanisms in F. kawagutii to cope with heat stress and phosphorus-deficiency stress. Their quantitative expression dynamics, however, requires further verification with triplicated experiments, and the data reported here only provide clues for generating testable hypotheses about molecular mechanisms underpinning responses and adaptation in F. kawagutii to temperature and nutrient stresses.

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Section: Discussionmentioning

confidence: 96%

Transcriptomic Responses to Thermal Stress and Varied Phosphorus Conditions in Fugacium kawagutii

Lin

Zhang

2019

Microorganisms

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“…In the qRT-PCR experiments, all 10 primer pairs had acceptable amplification efficiency, indicating that the qRT-PCR results are reliable. Stability analysis of candidate reference genes was mainly based on the analysis using geNorm software [ 52 – 54 ]. Combined with NormFinder and BestKeeper, the data for selecting an optimal reference gene was further supported.…”

Section: Discussionmentioning

confidence: 99%

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Zhao

Yang

Chen

et al. 2018

BioMed Research International

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Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC, and H2A, were tested for their adequacy in Lentinula edodes (L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of L. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm, TUB had the lowest M values in L. edodes strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was TUB (0.030), and the best candidate reference gene in strain 18N44 was UBI (0.047). In BestKeeper analysis, the standard deviation (SD) values of UBC, TUA, H2A, EF1, ACT, 18S, and GTP in strain 18 and those of GADPH and GTP in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only UBI and TUB were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, TUB was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in L. edodes mycelia under high-temperature stress.

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“…Evaluating the stability of pre-selected candidate reference genes using RT-qPCR is the most frequently used method for reference genes selection (Amil-Ruiz et al, 2013; Martins et al, 2016; Chong et al, 2017; Dzaki et al, 2017). However, this method is overwhelmingly dependent on the availability of pre-selected genes, and such a method may neglect the genes that would have been the most stable reference genes for a given experimental condition (Zhao et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi

Huang

Zhan

2019

Front. Genet.

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Rapid adaptation/accommodation to changing environments largely contributes to maximal survival of invaders during biological invasions, usually leading to success in crossing multiple barriers and finally in varied environments in recipient habitats. Gene expression is one of the most important and rapid ways during responses to environmental stresses. Selection of proper reference genes is the crucial prerequisite for gene expression analysis using the common approach, real-time quantitative PCR (RT-qPCR). Here we identified eight candidate novel reference genes from the RNA-Seq data in an invasive model ascidian Ciona savignyi under temperature and salinity stresses. Subsequently, the expression stability of these eight novel reference genes, as well as other six traditionally used reference genes, was evaluated using RT-qPCR and comprehensive tool RefFinder. Under the temperature stress, two traditional reference genes, ribosomal proteins S15 and L17 ( RPS15, RPL17 ), and one novel gene Ras homolog A ( RhoA ), were recommended as the top three stable genes, which can be used to normalize target genes with a high and moderate expression level, respectively. Under the salinity stress, transmembrane 9 superfamily member ( TMN ), MOB kinase activator 1A-like gene ( MOB ) and ubiquitin-conjugating enzyme ( UBQ2 ) were suggested as the top three stable genes. On the other hand, several commonly used reference genes such as α-tubulin ( TubA ), β-tubulin ( TubB ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) showed unstable expressions, thus these genes should not be used as internal controls for gene expression analysis. We also tested the expression level of an important stress response gene, large proline-rich protein bag6-like gene ( BAG ) using different reference genes. As expected, we observed different results and conclusions when using different normalization methods, thus suggesting the importance of selection of proper reference genes and associated normalization methods. Our results provide a valuable reference gene resource for the normalization of gene expression in the study of environmental adaptation/accommodation during biological invasions using C. savignyi as a model.

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Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

Cited by 16 publications

References 55 publications

Transcriptomic Responses to Thermal Stress and Varied Phosphorus Conditions in Fugacium kawagutii

Transcriptomic Responses to Thermal Stress and Varied Phosphorus Conditions in Fugacium kawagutii

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi

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