Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

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Reference Gene Selection for miRNA qRT-PCR Analysis in Lily

10.21203/rs.3.rs-102523/v1 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Xue Gao ◽

Lian-Juan Wang ◽

Yu-Qian Zhao ◽

Gui-Xia Jia

Keyword(s):

Reference Gene ◽

Stress Responses ◽

Reference Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Critical Element ◽

Biotic And Abiotic Stress ◽

Experimental Conditions ◽

Qrt Pcr

Abstract Background: The selection of reliable reference genes is a critical element for obtaining accurate gene expression data to assess quantitative real-time polymerase chain reaction (qRT-PCR) performance. It is critical to use suitable reference genes in miRNA qRT-PCR because of short amplification products and large differences in the expression levels of target miRNAs involved in some biological processes. However, in lily, which exhibits a large complex genome but lacks a reference, the available miRNA reference genes for use in qRT-PCR under various treatment conditions are limited, and their reliability has rarely been systematically evaluated.Results: In this study, 8 candidate reference genes, including three classic housekeeping genes and five potential miRNAs from the miRNA library of L. × formolongi, were selected and assessed for expression stability utilizing the BestKeeper, geNorm and Normfinder tools, together with the Delta Ct method, across a diverse set of biotic and abiotic experimental conditions (developmental stages, tissues, heat stress and pathogen defence) to determine the best reference gene(s) for L. × formolongi and L. regale. The final ranking was reordered by using RankAggreg, and the results showed that the novel miRNA PC-3p-67_108977 and the conserved miRNAs miR399a, miR399a and U6 were the most stable genes for L. × formolongi and L. regale, respectively, under all tested experimental conditions. Additionally, PC-3p-67_108977 and U6 were the most suitable genes for qRT-PCR studies in lily.Conclusions: This study provides a comprehensive evaluation of the reliability of reference genes for miRNA studies on development and biotic and abiotic stress responses in different lilies. These results will be beneficial for miRNA identification and functional studies of lilies in the future.

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Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽

10.3390/genes12070960 ◽

2021 ◽

Vol 12 (7) ◽

pp. 960

Author(s):

Meagan Archer ◽

Jianping Xu

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Specific Method ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Physiological Processes ◽

The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

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Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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

Frontiers in Physiology ◽

10.3389/fphys.2021.669510 ◽

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.

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Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

Frontiers in Physiology ◽

10.3389/fphys.2021.752768 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gothandapani Sellamuthu ◽

Shan Amin ◽

Jan Bílý ◽

Jirí Synek ◽

Roman Modlinger ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Gene Selection ◽

Experimental Conditions ◽

Tissue Specific ◽

Reference Gene Selection ◽

Ips Sexdentatus

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

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Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

PLoS ONE ◽

10.1371/journal.pone.0053006 ◽

2013 ◽

Vol 8 (1) ◽

pp. e53006 ◽

Cited By ~ 99

Author(s):

Rumei Li ◽

Wen Xie ◽

Shaoli Wang ◽

Qingjun Wu ◽

Nina Yang ◽

...

Keyword(s):

Reference Gene ◽

Bemisia Tabaci ◽

Gene Selection ◽

Pcr Analysis ◽

Sweetpotato Whitefly ◽

Reference Gene Selection ◽

Qrt Pcr ◽

Selection For

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Reference gene selection for qRT-PCR analysis of flower development in Lagerstroemia indica and L. speciosa

PLoS ONE ◽

10.1371/journal.pone.0195004 ◽

2018 ◽

Vol 13 (3) ◽

pp. e0195004 ◽

Cited By ~ 12

Author(s):

Tangchun Zheng ◽

Zhilin Chen ◽

Yiqian Ju ◽

Han Zhang ◽

Ming Cai ◽

...

Keyword(s):

Reference Gene ◽

Flower Development ◽

Gene Selection ◽

Pcr Analysis ◽

Reference Gene Selection ◽

Qrt Pcr ◽

Selection For

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Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

International Journal of Molecular Sciences ◽

10.3390/ijms23020738 ◽

2022 ◽

Vol 23 (2) ◽

pp. 738

Author(s):

Xiu-Mei Dong ◽

Wei Zhang ◽

Shi-Bao Zhang

Keyword(s):

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Molecular Mechanisms ◽

Gene Selection ◽

Expression Profiles ◽

Flower Color ◽

Pcr Analysis ◽

Color Formation ◽

Qrt Pcr

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.

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Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

Frontiers in Physiology ◽

10.3389/fphys.2021.663338 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mengyi Li ◽

Xinan Li ◽

Chao Wang ◽

Qiuchi Li ◽

Saige Zhu ◽

...

Keyword(s):

Reference Genes ◽

Developmental Stages ◽

Expression Profiles ◽

Rhopalosiphum Padi ◽

Pcr Analysis ◽

Future Research ◽

Wing Dimorphism ◽

Experimental Conditions ◽

Online Tool ◽

Qrt Pcr

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

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Identification of the Most Suitable Reference Gene for Gene Expression Studies With Development and Abiotic Stress Response in Bromus Sterilis

10.21203/rs.3.rs-156798/v1 ◽

2021 ◽

Author(s):

Madhab Kumar Sen ◽

Katerina Hamouzova ◽

Pavlina Kosnarova ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

Abstract Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide-resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can contribute to elucidation of the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes were evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used softwares for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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