Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

Abstract The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

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Evaluation of qPCR Reference Genes For Taimen (Hucho Taimen)

10.21203/rs.3.rs-646522/v1 ◽

2021 ◽

Author(s):

Xiaoxing Yang ◽

Guangxiang Tong ◽

Le Dong ◽

Guopan Tang ◽

Yongquan Zhang ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Reference Genes ◽

28S Rrna ◽

Experimental Conditions ◽

Stress Genes ◽

Qrt Pcr ◽

Processing Times ◽

Reliable Quantification ◽

Hucho Taimen

Abstract As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquacultural research. Understanding the biology of taimen (Hucho taimen) has attracted increased interest because of its ecological and economic values. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimised for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using four methods, geNorm, NormFinder, BestKeeper and RefFinder. The expression levels of the 10 genes were detected using samples from 48 experimental groups that consisted of six tissues (blood, heart, brain, gill, skin and liver) collected under five heat-stress conditions (18, 20, 22, 24 and 26°C) and four heat-stress processing times (4, 24, 48 and 96 h). RPS29 and RPL19 were the most stable genes among all the samples, whereas 28S rRNA, ARBPR and 18S rRNA were the least stable. These results were verified by an expression analysis of the heat-stress genes (hsp60 and hsp70) of taimen. In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions, and these results allow the reliable study of gene expression in taimen.

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Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress

International Journal of Medicinal Mushrooms ◽

10.1615/intjmedmushrooms.2017024487 ◽

2017 ◽

Vol 19 (11) ◽

pp. 1029-1040 ◽

Cited By ~ 3

Author(s):

Yong-Nan Liu ◽

Xiao-Xiao Lu ◽

Ang Ren ◽

Liang Shi ◽

Ai-Liang Jiang ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Heat Shock ◽

Heat Shock Protein ◽

Reference Genes ◽

Ganoderma Lucidum ◽

Protein Gene ◽

Medicinal Mushroom ◽

Heat Shock Protein Gene

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Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae)

10.7287/peerj.preprints.662 ◽

2014 ◽

Author(s):

Chunxiao Chunxiao Yang ◽

Hui Li ◽

Huipeng Pan ◽

Yabin Ma ◽

Deyong Zhang ◽

...

Keyword(s):

Gene Expression ◽

Heat Shock ◽

Heat Shock Protein ◽

Ribosomal Rna ◽

Expression Profiles ◽

Housekeeping Genes ◽

Pcr Analysis ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative real-time PCR (qRT-PCR) is a powerful technique for measuring and evaluating gene expressions during different biological processes. To facilitate gene expression studies, normalization with respect to stable housekeeping genes (HKGs) is mandatory. The western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), the main vector of Tomato spotted wilt virus (TSWV), is a very destructive invasive species. In this study, expression profiles of 11 candidate HKGs, including β-actin (Actin), α-tubulin (Tubulin), elongation factor 1 α (EF1A), vacuolar-typeH+-ATPase (ATPase), NADH-ubiquinone oxidoreductase (NADH), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), ribosomal protein l32 (RPL32), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S), from no nviruliferous and viruliferous F. occidentalis were investigated. Four distinct algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to determine the performance of these genes as endogenous controls under the virus condition. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidates, HSP70 , HSP60, EF1A, and RPL32 were the most stable housekeeping genes. This work is the initial first step to establish a standardized qRT-PCR analysis in F. occidentalis. Additionally, this study lays a foundation for the research in the interactions between TSWV and F. occidentalis.

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Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae)

10.7287/peerj.preprints.662v1 ◽

2014 ◽

Author(s):

Chunxiao Chunxiao Yang ◽

Hui Li ◽

Huipeng Pan ◽

Yabin Ma ◽

Deyong Zhang ◽

...

Keyword(s):

Gene Expression ◽

Heat Shock ◽

Heat Shock Protein ◽

Ribosomal Rna ◽

Expression Profiles ◽

Housekeeping Genes ◽

Pcr Analysis ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative real-time PCR (qRT-PCR) is a powerful technique for measuring and evaluating gene expressions during different biological processes. To facilitate gene expression studies, normalization with respect to stable housekeeping genes (HKGs) is mandatory. The western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), the main vector of Tomato spotted wilt virus (TSWV), is a very destructive invasive species. In this study, expression profiles of 11 candidate HKGs, including β-actin (Actin), α-tubulin (Tubulin), elongation factor 1 α (EF1A), vacuolar-typeH+-ATPase (ATPase), NADH-ubiquinone oxidoreductase (NADH), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), ribosomal protein l32 (RPL32), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S), from no nviruliferous and viruliferous F. occidentalis were investigated. Four distinct algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to determine the performance of these genes as endogenous controls under the virus condition. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidates, HSP70 , HSP60, EF1A, and RPL32 were the most stable housekeeping genes. This work is the initial first step to establish a standardized qRT-PCR analysis in F. occidentalis. Additionally, this study lays a foundation for the research in the interactions between TSWV and F. occidentalis.

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Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽

10.3390/f11020193 ◽

2020 ◽

Vol 11 (2) ◽

pp. 193

Author(s):

Dandan Li ◽

Sen Yu ◽

Minzhen Zeng ◽

Xiao Liu ◽

Jia Yang ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Pcr Analysis ◽

Stable Gene ◽

Larix Olgensis ◽

Qrt Pcr ◽

Study Gene Expression ◽

Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

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Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Biology Methods and Protocols ◽

10.1093/biomethods/bpw005 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Roshini Kalagara ◽

Weimin Gao ◽

Honor L. Glenn ◽

Colleen Ziegler ◽

Laura Belmont ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Stable Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

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Mitigation of Heat Stress in Broiler Chickens with Heat Shock Protein 70 Gene Expression as its Indicator

Indonesian Bulletin of Animal and Veterinary Sciences ◽

10.14334/wartazoa.v30i4.2563 ◽

2020 ◽

Vol 30 (4) ◽

pp. 177

Author(s):

Cecep Hidayat ◽

Komarudin . ◽

E Wina

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Heat Shock ◽

Heat Shock Protein ◽

Broiler Chickens ◽

Broiler Chicken ◽

Production Performance ◽

Hsp70 Gene ◽

Hsp 70 ◽

Manganese Chromium

<p class="awabstrak2"><span lang="EN-US">Heat stress is an important issue in broiler chicken farms in tropical countries, such as Indonesia. Heat stress is very detrimental to broiler chickens because reducing production performance, health, and causing mortality. In the condition of heat stress, broilers synthesize Heat Shock Protein (HSP) quickly as the body's response to heat stress. HSP 70 is the most studied HSP group related to heat stress. The objective of this study was to review the nutritional approach that has been done to mitigate heat stress in broiler chickens with the HSP70 gene expression as its indicator. Based on some studies, nutritional approaches that can be taken are through the management of feed availability, supplementation of vitamin C, vitamin E, plant bioactives, amino acids (taurine and glutamine), probiotics, prebiotics, synbiotics, manan oligo saccharide (MOS) and minerals (selenium, zinc, manganese, chromium). By these approaches, HSP70 gene expression decreased indicating that the heat stress level of broiler chicken also reduced. It can be concluded that the nutritional approach can be used as a method for heat stress mitigation in broilers with the HSP70 gene expression indicator. </span></p>

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