reference genes
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Insects ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 97
Xudong Zhao ◽  
Yishu Geng ◽  
Tianyi Hu ◽  
Yongang Zhao ◽  
Suling Yang ◽  

The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

2022 ◽  
Vol 12 ◽  
Ningning Fu ◽  
Jiaxing Li ◽  
Ming Wang ◽  
Lili Ren ◽  
Shixiang Zong ◽  

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

2022 ◽  
Vol 12 ◽  
Haiyan Fu ◽  
Tubiao Huang ◽  
Cheng Yin ◽  
Zhenhua Xu ◽  
Chao Li ◽  

Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

2022 ◽  
Vol 12 (1) ◽  
Xiaoxing Yang ◽  
Guangxiang Tong ◽  
Le Dong ◽  
Ting Yan ◽  
Huan Xu ◽  

AbstractAs a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

2022 ◽  
Vol 23 (2) ◽  
pp. 738
Xiu-Mei Dong ◽  
Wei Zhang ◽  
Shi-Bao Zhang

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

2022 ◽  
Zhi-Peng Zhu ◽  
Jian-Xiang Yu ◽  
Ke-Xin Wu ◽  
Qin-Yi Xu ◽  
Yi-Jun Kang ◽  

Abstract Baishouwu (Cynanchum auriculatum) is a kind of critical Chinese herbal medicine. However, compared with the studies of other Chinese herbal medicines, the screening study on the reference genes of C. auriculatum is still the blank. Deterioration of the natural environment severely affects the growth and development of C. auriculatum. This study screened and identified suitable reference genes of C. auriculatum under various stress conditions. Based on qRT-PCR, geNorm, NormFinder, BestKeeper, and RefFinder were used for the expression stability evaluation of 12 potential reference genes from C. auriculatum. The ranking table showed that optimal reference genes included EF2 and SAMDC (heat stress), CYP and TUB-β (cold stress), TUB-α and GAPDH (drought stress), SAMDC and TUB-α (waterlogging stress), along with EF2 and ACT7 (salt stress). These results also demonstrated that under different abiotic stresses, suitable reference genes of plants should be selected for qRT-PCR analysis.

2022 ◽  
Vol 12 (1) ◽  
Kui-Peng Li ◽  
Wei Li ◽  
Gui-Yun Tao ◽  
Kai-Yong Huang

AbstractThe radial change (RC) of tree stem is the process of heartwood formation involved in complex molecular mechanism. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), an evergreen species, is an important fast-growing timber tree in southern China. In this study, the top four stable genes (IDH, UBC2, RCA and H2B) were selected in RC tissues of 15 years old Chinese fir stem (RC15) and the genes (H2B, 18S, TIP41 and GAPDH) were selected in RC tissues of 30 years old Chinese fir stem (RC30). The stability of the reference genes is higher in RC30 than in RC15. Sixty-one MYB transcripts were obtained on the PacBio Sequel platform from woody tissues of one 30 years old Chinese fir stem. Based on the number of MYB DNA-binding domain and phylogenetic relationships, the ClMYB transcripts contained 21 transcripts of MYB-related proteins (1R-MYB), 39 transcripts of R2R3-MYB proteins (2R-MYB), one transcript of R1R2R3-MYB protein (3R-MYB) belonged to 18 function-annotated clades and two function-unknown clades. In RC woody tissues of 30 years old Chinese fir stem, ClMYB22 was the transcript with the greatest fold change detected by both RNA-seq and qRT-PCR. Reference genes selected in this study will be helpful for further verification of transcript abundance patterns during the heartwood formation of Chinese fir.

2021 ◽  
Vol 12 (1) ◽  
pp. 153
Lynsey Steel ◽  
David M. Ansell ◽  
Enrique Amaya ◽  
Sarah H. Cartmell

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

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