Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa)

BackgroundThe veined rapa whelkRapana venosais an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression inR. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes inR. venosafor use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α(EF-1α),α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1αsubcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH),β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found thatEF-1αwas the most stable internal control gene in almost all adult tissue samples investigated withRL5andRL28as secondary choices. For the normalization of a single specific tissue, we suggested thatEF-1αandNDUFA7are the best combination in gonad, as well asCOX1andRL28for intestine,EF-1αandRL5for kidney,EF-1αandCOX1for gill,EF-1αandRL28for Leiblein and mantle,EF-1α,RL5, andNDUFA7for liver, GAPDH,PPIA, andRL28for hemocyte. From a developmental perspective, we found thatRL28was the most stable gene in all developmental stages measured, andCOX1andRL5were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization,PPIA,RS25, andRL28for stage 1,RL5andRL28for stage 2 and 5,RL28andNDUFA7for stage 3, andPPIAandTUBBfor stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development ofR. venosain the future.

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Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

10.1101/499186 ◽

2018 ◽

Author(s):

Zhezhi Wang ◽

Wen Zhou ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Housekeeping Genes ◽

Potential Candidate ◽

Qrt Pcr ◽

Wide Range ◽

Different Tissues

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-β were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-α were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

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Peer Review #2 of "Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa) (v0.1)"

10.7287/peerj.3398v0.1/reviews/2 ◽

2017 ◽

Author(s):

MLY Bangaru

Keyword(s):

Peer Review ◽

Housekeeping Genes ◽

Internal Controls ◽

Pcr Analysis ◽

Rt Pcr ◽

Rapana Venosa ◽

Selection Of ◽

Rapa Whelk

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Peer Review #3 of "Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa) (v0.1)"

10.7287/peerj.3398v0.1/reviews/3 ◽

2017 ◽

Author(s):

S Kuchipudi

Keyword(s):

Peer Review ◽

Housekeeping Genes ◽

Internal Controls ◽

Pcr Analysis ◽

Rt Pcr ◽

Rapana Venosa ◽

Selection Of ◽

Rapa Whelk

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Peer Review #1 of "Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa) (v0.1)"

10.7287/peerj.3398v0.1/reviews/1 ◽

2017 ◽

Keyword(s):

Peer Review ◽

Housekeeping Genes ◽

Internal Controls ◽

Pcr Analysis ◽

Rt Pcr ◽

Rapana Venosa ◽

Selection Of ◽

Rapa Whelk

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Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Scientific Reports ◽

10.1038/s41598-020-79298-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuping Li ◽

Xiaoju Liang ◽

Xuguo Zhou ◽

Yu An ◽

Ming Li ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stage ◽

Reference Genes ◽

Developmental Stages ◽

Individual Factors ◽

Congeneric Species ◽

Spatio Temporal ◽

The Stability ◽

Different Tissues ◽

Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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Evaluation of Appropriate Reference Genes For Investigating Gene Expression in Chlorops oryzae (Diptera: Chloropidae)

Journal of Economic Entomology ◽

10.1093/jee/toz142 ◽

2019 ◽

Vol 112 (5) ◽

pp. 2207-2214 ◽

Cited By ~ 1

Author(s):

Ping Tian ◽

Lin Qiu ◽

Ailin Zhou ◽

Guo Chen ◽

Hualiang He ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Genes ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Quantitative Polymerase Chain Reaction ◽

Housekeeping Genes ◽

Future Research ◽

Economic Damage ◽

Physiological Processes

Abstract Reverse transcription quantitative polymerase chain reaction (PCR) has become an invaluable technique for analyzing gene expression in many insects. However, this approach requires the use of stable reference genes to normalize the data. Chlorops oryzae causes significant economic damage to rice crops throughout Asia. The lack of suitable reference genes has hindered research on the molecular mechanisms underlying many physiological processes of this species. In this study, we used quantitative real-time PCR to evaluate the expression of eight C. oryzae housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (βACT), beta-tubulin (βTUB), Delta Elongation factor-1 (EF1δ), ribosomal protein S11 (RPS11), RPS15, C-terminal-Binding Protein (CtBP), and ribosomal protein 49 (RP49) in different developmental stages and tissues in both larvae and adults. We analyzed the data with four different software packages: geNorm, NormFinder, BestKeeper, and RefFinder and compared the results obtained with each method. The results indicate that PRS15 and RP49 can be used as stable reference genes for quantifying gene expression in different developmental stages and larval tissues. GAPDH and βACT, which have been considered stable reference genes by previous studies, were the least stable of the candidate genes with respect to larval tissues. GAPDH was, however, the most stable reference gene for adult tissues. We verified the candidate reference genes identified and found that the expression levels of Cadherins (Cads) changed when different reference genes were used to normalize gene expression. This study provides a valuable foundation for future research on gene function, and investigating the molecular basis of physiological processes, in C. oryzae.

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Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

PeerJ ◽

10.7717/peerj.7133 ◽

2019 ◽

Vol 7 ◽

pp. e7133 ◽

Cited By ~ 4

Author(s):

Wen Zhou ◽

Shiqiang Wang ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Pcr Analysis ◽

Potential Candidate ◽

Qrt Pcr ◽

Expression Studies ◽

Different Tissues

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.

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Validation of Stable Housekeeping Genes for Quantitative Real Time PCR in Golden Syrian Hamster

Indian Journal of Animal Research ◽

10.18805/ijar.b-1237 ◽

2020 ◽

Author(s):

Jin-xin Miao ◽

Jian-yao Wang ◽

Nick Robl ◽

Hao-ran Guo ◽

Shao-he Song ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Real Time ◽

Syrian Hamster ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Pancreatic Tissue ◽

Golden Syrian Hamster ◽

The Stability ◽

Selection Of

Background: Golden Syrian hamster (GSH) have many advantages as animal models in preclinical research, but their application is currently limited by the lack of standardized techniques for analyzing gene expression. Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for analyzing gene expression, but its reliability depends upon the selection of stable reference (“housekeeping”) genes for proper normalization. The current study was aimed to RT-qPCR for investigated to determine the stability housekeeping gene in golden Syrian hamster.Methods: During the period of June 2019 to October 2019, the expression stability of eight commonly-used housekeeping genes (glyceraldehyde-3phosphate dehydrogenase, Gapdh; b-actin, Actb; hypoxanthine phosphoribosyl transferase 1, Hprt1; ribosomal protein L13a, Rpl13a; ribosomal protein S18, Rps18; Beta-2-microglobulin, B2m; Tubulin beta class I, Tubb; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta, Ywhaz) were investigated in lung, liver, spleen and pancreatic tissue of golden Syrian hamsters using BestKeeper, geNorm, NormFinder software, comparative delta Ct method and RefFinder. Result: It was found that the stability of gene expression varied among tissue where in Actb was the most stably expressed housekeeping gene for lung tissue, Hprt1 for liver, Rpl13a for spleen and Tubb for pancreatic tissue. In contrast, the least stable housekeeping gene in both liver and spleen was Gapdh, Rps18 in pancreas and Ywhaz in lung tissue. These data provide a critical evaluation of housekeeping genes in GSH tissues that can be used as a guide for selection of the appropriate genes in future studies.

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