Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in Apolygus lucorum (Hemiptera: Miridae)

2019 ◽  
Vol 113 (1) ◽  
pp. 451-460
Author(s):  
Jing Luo ◽  
Aoli Wang ◽  
Yanxia Cheng ◽  
Haoling Rong ◽  
Libin Guo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Large Scale ◽  
Control Strategies ◽  
Quantitative Polymerase Chain Reaction ◽  
Life Stage ◽  
Genetic Studies ◽  
Apolygus Lucorum ◽  
Economic Significance ◽  
Stability Ranking ◽  
Suitable Reference

Abstract Apolygus lucorum (Meyer-Dür) is a destructive pest to >280 plants. Major economic significance and pesticide resistance issues have created a need for integrated pest management (e.g., RNAi, entomopathogen-based bioinsecticides) for A. lucorum. To better develop these control strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been no reports on appropriate reference genes in A. lucorum. Here, we evaluated nine widely utilized reference genes including EF1γ, RPL32, RPL27, SDH, TBP, ACT, ACT2, GAPDH, and βTUB for their expression stabilities in A. lucorum under five different conditions i.e., life stage, tissue, sex, dsRNA injection, and entomopathogen infection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend RPL27 and RPL32 as the most appropriate reference genes for molecular studies in different life stages and tissues; GAPDH and EF1γ for different sexes and entomopathogen infection studies; and RPL27 and EF1γ for RNAi studies. The results of this study will help improve the accuracy and reliability for normalizing the RT-qPCR data for further molecular analysis in A. lucorum.

scholarly journals Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae)

Insects ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 359
Author(s):  
Xinhua Fu ◽  
Victor Benno Meyer-Rochow
Keyword(s):  
Reference Genes ◽  
Communication Systems ◽  
Large Scale ◽  
Quantitative Polymerase Chain Reaction ◽  
Life Stage ◽  
Research Strategies ◽  
Genetic Studies ◽  
Stability Ranking ◽  
Polymerase Chain ◽  
Suitable Reference

Aquatica leii Fu and Ballantyne is a species of rare aquatic firefly and endemic in China. It is considered good material to study the molecular mechanism of sexual flash communication systems. To improve conservation and behavioral research strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been very few reports on appropriate reference genes in any species of firefly. Here, we evaluated eight widely utilized reference genes including 18S, Actin, Reep5, Odc1, Tub, Gapdh, Ef1a and S27Ae for their expression stabilities in A. leii under three different conditions, i.e., life stage, tissue and dsRNA injection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend S27Ae and Reep5 as the most appropriate reference genes for molecular studies in different life stages; Ef1a and Odc1 for different tissues; Tub and Odc1 for RNAi studies. The most appropriate reference genes in all treatments are S27Ae and Tub. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis.

scholarly journals Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone Flaccida Fr. Schmidt at Different Growing Stages

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 459
Author(s):  
Zeying Zhao ◽  
Hanwen Zhou ◽  
Zhongnan Nie ◽  
Xuekui Wang ◽  
Biaobiao Luo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Cultivated Plants ◽  
Gene Expressions ◽  
Natural Habitats ◽  
Triterpenoid Saponins ◽  
Qpcr Normalization ◽  
Suitable Reference

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

scholarly journals Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

2021 ◽  
Vol 6 ◽  
pp. 197
Author(s):  
John C.W. Hildyard ◽  
Dominic J. Wells ◽  
Richard J. Piercy
Keyword(s):  
Gene Expression ◽  
Mouse Embryo ◽  
Reference Genes ◽  
Developmental Regulation ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Cell Types ◽  
Late Gestation ◽  
Developmental Expression ◽  
Suitable Reference

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5–E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes (ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall. HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable (CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

scholarly journals Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiaoxin Xie ◽  
Tinghui Liu ◽  
Adel Khashaveh ◽  
Chaoqun Yi ◽  
Xiaoxu Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Qpcr Analysis ◽  
Hippodamia Variegata ◽  
Convenient Technique ◽  
Suitable Reference

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

scholarly journals Transcriptome-wide identification and evaluation of optimal reference genes for RT-qPCR expression analysis of Saccharina latissima responses to biotic and abiotic stress

2020 ◽  
Author(s):  
Qikun Xing ◽  
Sylvie Rousvoal ◽  
Catherine Leblanc
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Reference Genes ◽  
Large Scale ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Light Stress ◽  
Saccharina Latissima ◽  
Biotic And Abiotic Stress ◽  
Intertidal Environment

AbstractSaccharina latissima, known as sugar kelp, is a brown macroalga with huge ecological and economic values. In marine intertidal environment, S. latissima has to cope with both biotic and abiotic stress, which can cause the reduction of the yield during cultivation. To better understand the physiological responses of S. latissima under different stress conditions, large-scale transcriptomic analyses are useful to explore global metabolic pathway regulations. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful and rapid method for further quantifying changes in gene expression, and for targeting specific defense-related gene pathways. However, its level of accuracy is highly related to the expression stability of reference genes used for normalization and those still need to be evaluated in S. latissima. In this study, we therefore experimentally tested eight candidate reference genes identified from in silico screening of public transcriptomic datasets of S. latissima from different abiotic and biotic stress treatments. The stability analysis using complementary statistical approaches showed that EIF5B and ATPase are the most stable reference genes under biotic stress, whereas, under temperature and light stress, their combination with NDH gene is the best choice for RT-qPCR normalization. The validated reference genes were used to monitor the expression of target genes, related to oxidative responses, such as those involved in oxylipin pathways, in S. latissima plantlets submitted to different stress in laboratory-controlled conditions.

scholarly journals Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

2020 ◽  
Vol 21 (23) ◽  
pp. 9195
Author(s):  
Johannes Hasler ◽  
Luan Phelipe Hatt ◽  
Martin James Stoddart ◽  
Angela Rita Armiento
Keyword(s):  
Gene Expression ◽  
Osteogenic Differentiation ◽  
Reference Genes ◽  
High Throughput Screening ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Phenotype ◽  
Research Fields ◽  
Qpcr Analysis ◽  
The Stability ◽  
Suitable Reference

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.

scholarly journals Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

Forests ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 441 ◽  
Author(s):  
Zhonghua Tu ◽  
Ziyuan Hao ◽  
Weiping Zhong ◽  
Huogen Li
Keyword(s):  
Reference Genes ◽  
Target Gene ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Stability ◽  
Chain Reaction ◽  
Multiple Organs ◽  
Liriodendron Chinense ◽  
Polymerase Chain ◽  
The Difference ◽  
Suitable Reference

The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.

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