Analysis of the stability of housekeeping gene expression in the left cardiac ventricle of rats submitted to chronic intermittent hypoxia

Julian, Guilherme Silva; Oliveira, Renato Watanabe de; Tufik, Sérgio; Chagas, Jair Ribeiro

doi:10.1590/s1806-37562015000000133

Cited by 11 publications

(11 citation statements)

References 20 publications

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“…When all expression stability values were integrated from the five body parts, the stability rank from the most to least stable was as follows: RAD1a > RPS18 > RPS5 > ARF1 >> GAPDH ( Table 1 and Fig 2A ). Considering a stability value < 0.15 as the criterion for suitable reference gene selection in NormFinder [ 57 , 58 ], all genes were acceptable across different conditions, but GAPDH (stability value = 0.166) was not suggested as a qRT-PCR reference gene for thorax of honey bees exposed to pesticides ( Table 1 and Fig 2A ).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to RefFinder, three software also analyzed that RAD1a and RPS18 were ranked as the most stable ( Fig 2 ). Based on the criteria: mean stability < 0.15 in NormFinder [ 57 , 58 ], SD value < 1.0 in BestKeeper [ 59 ], and M < 0.5 in geNorm [ 60 , 61 ], the calculated values of both genes were below the cut-off values ( Fig 2 and Tables 1 and S2 – S4 ). C q distribution analysis also revealed CV values < 1, which indicates low variance ( Fig 1 ) [ 56 ], suggesting that the combination of RAD1a and RPS18 can be selected as reference genes for target gene normalization in different body parts from honey bees exposed to various pesticides.…”

Section: Discussionmentioning

confidence: 99%

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Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera

Kim

Cha

et al. 2022

PLoS ONE

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Recently, pesticides have been suggested to be one of the factors responsible for the large-scale decline in honey bee populations, including colony collapse disorder. The identification of the genes that respond to pesticide exposure based on their expression is essential for understanding the xenobiotic detoxification metabolism in honey bees. For the accurate determination of target gene expression by quantitative real-time PCR, the expression stability of reference genes should be validated in honey bees exposed to various pesticides. Therefore, in this study, to select the optimal reference genes, we analyzed the amplification efficiencies of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAD1a) and their expression stability values using four programs (geNorm, NormFinder, BestKeeper, and RefFinder) across samples of five body parts (head, thorax, gut, fat body, and carcass) from honey bees exposed to seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin). Among these five candidate genes, a combination of RAD1a and RPS18 was suggested for target gene normalization. Subsequently, expression levels of six genes (AChE1, CYP9Q1, CYP9Q2, CYP9Q3, CAT, and SOD1) were normalized with a combination of RAD1a and RPS18 in the different body parts from honey bees exposed to pesticides. Among the six genes in the five body parts, the expression of SOD1 in the head, fat body, and carcass was significantly induced by six pesticides. In addition, among seven pesticides, flupyradifurone statistically induced expression levels of five genes in the fat body.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera

Kim

Cha

et al. 2022

PLoS ONE

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“…Considering the cutoff values in each algorithm, most of the seven candidate genes could be deemed acceptable for use as reference genes when analysing gene expression in different conditions in honey bees. Although most previous studies did not set a cutoff value for gene stability in NormFinder analysis 6 – 8 , 14 – 16 , 21 , 22 , several recent studies have suggested values < 0.15 as a suitable cutoff 20 , 26 , 27 . Based on this criterion, all seven genes were suitable reference genes according to NormFinder.…”

Section: Discussionmentioning

confidence: 99%

Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

Jeon

Moon

Kim

et al. 2020

Sci Rep

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Honey bees are both important pollinators and model insects due to their highly developed sociality and colony management. To better understand the molecular mechanisms underlying honey bee colony management, it is important to investigate the expression of genes putatively involved in colony physiology. Although quantitative real-time PCR (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different conditions) must first be identified to ensure accurate normalisation of target genes. To identify reliable reference genes in honey bee (Apis mellifera) colonies, therefore, we evaluated seven candidate genes (ACT , EIF, EF1, RPN2, RPS5, RPS18 and GAPDH) in samples collected from three honey bee tissue types (head, thorax and abdomen) across all four seasons using three analysis programmes (NormFinder, BestKeeper and geNorm). Subsequently, we validated various normalisation methods using each of the seven reference genes and a combination of multiple genes by calculating the expression of catalase (CAT). Although the genes ranked as the most stable gene were slightly different on conditions and analysis methods, our results suggest that RPS5, RPS18 and GAPDH represent optimal honey bee reference genes for target gene normalisation in qRT-PCR analysis of various honey bee tissue samples collected across seasons. The Western honey bee, Apis mellifera L., plays an important role as a pollinator 1. In addition, the honey bee is considered to be a key model insect due to its relatively complex behaviours, including sociality, labour division and colony management 2. Previous studies have demonstrated that endocrine system status and gene expression are important factors for flexible honey bee colony management, which involves colonies seasonally regulating their labour division and population dynamics 3-5. In order to extend our understanding of the molecular mechanisms that underlie the regulation of honey bee colony physiology, information on the physiological functions of the genes putatively associated with colony management can be determined by analysing their expression profiles among different seasons and honey bee tissues 6,7. In quantitative real-time PCR (qRT-PCR), gene-specific mRNA (or cDNA) is quantified; this method has been used extensively because of its relative speed, sensitivity, replicability and accuracy 8,9. Therefore, qRT-PCR would be an ideal method for analysing the expression patterns of honey bee genes putatively involved in the plasticity of colony molecular physiology in samples collected across different seasons and tissues. However, because qRT-PCR results are highly sensitive to the initial amount of RNA content in the amplification reaction, the interpretation of target gene expression levels among various conditions would result in appreciable errors without the use of a reliable internal standard 7-10. Therefore, prior to analysing target gene expression levels among conditions, reference genes are requir...

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“…The set of customized assays employed to amplify the genes, namely primers/probes and cycle threshold (Ct) values, are described in Table 1. Expression values were normalized using three different endogenous controls ( beta ‐ actin , GAPDH , and B2 M ), as previously validated by our group for use with the IH protocol (Julian et al, 2016).…”

Section: Methodsmentioning

confidence: 99%

Intermittent hypoxia changes the interaction of the kinin–VEGF system and impairs myocardial angiogenesis in the hypertrophic heart

et al. 2021

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Analysis of the stability of housekeeping gene expression in the left cardiac ventricle of rats submitted to chronic intermittent hypoxia

Cited by 11 publications

References 20 publications

Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera

Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera

Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

Intermittent hypoxia changes the interaction of the kinin–VEGF system and impairs myocardial angiogenesis in the hypertrophic heart

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