Evaluation of Candidate Reference Genes Stability for Gene Expression Analysis by Reverse Transcription QPCR in Clostridium Perfringens

Toxin Gene ◽

Rrna Gene ◽

Experimental Conditions ◽

Abstract Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase+. These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-hour period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.

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 ◽

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.

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

Genes ◽

10.3390/genes11020124 ◽

2020 ◽

Vol 11 (2) ◽

pp. 124 ◽

Cited By ~ 1

Author(s):

Zhaoping Zhang ◽

Changjian Li ◽

Junqing Zhang ◽

Fang Chen ◽

Yongfu Gong ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Elongation Factor ◽

Papaver Somniferum ◽

Protein Subunit ◽

Experimental Conditions ◽

Analgesic Drugs

Papaver somniferum L. is an important medical plant that produces analgesic drugs used for the pain caused by cancers and surgeries. Recent studies have focused on the expression genes involved in analgesic drugs biosynthesis, and the real-time quantitative polymerase chain reaction (RT-qPCR) technique is the main strategy. However, no reference genes have been reported for gene expression normalization in P. somniferum. Herein, nine reference genes (actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin 2 (CYP2), elongation factor 1-alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic (GAPC2), nuclear cap-binding protein subunit 2 (NCBP2), protein phosphatase 2A (PP2A), TIP41-like protein (TIP41), and tubulin beta chain (TUB)) of P. somniferum were selected and analyzed under five different treatments (cold, drought, salt, heavy metal, and hormone stress). Then, BestKeeper, NormFinder, geNorm, and RefFinder were employed to analyze their gene expression stability. The results reveal that NCBP2 is the most stable reference gene under various experimental conditions. The work described here is the first report regarding on reference gene selection in P. somniferum, which could be used for the accurate normalization of the gene expression involved in analgesic drug biosynthesis.

Regulation of toxin gene expression in Clostridium perfringens

Research in Microbiology ◽

10.1016/j.resmic.2014.09.010 ◽

2015 ◽

Vol 166 (4) ◽

pp. 280-289 ◽

Cited By ~ 20

Author(s):

Kaori Ohtani ◽

Tohru Shimizu

Keyword(s):

Gene Expression ◽

Clostridium Perfringens ◽

Toxin Gene ◽

Quantification of Cell Proliferation and Alpha-Toxin Gene Expression of Clostridium perfringens in the Development of Necrotic Enteritis in Broiler Chickens

Applied and Environmental Microbiology ◽

10.1128/aem.01108-07 ◽

2007 ◽

Vol 73 (21) ◽

pp. 7110-7113 ◽

Cited By ~ 35

Author(s):

Weiduo Si ◽

Joshua Gong ◽

Yanming Han ◽

Hai Yu ◽

John Brennan ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Regression Model ◽

Clostridium Perfringens ◽

Broiler Chickens ◽

Toxin Gene ◽

Necrotic Enteritis ◽

Lesion Score ◽

Alpha Toxin ◽

ABSTRACT Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b 0 + b 1 X − b 2 X 2. The average C. perfringens count of 5 log10 CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.

Identification and Validation of Reference Genes for Gene Expression Analysis in Different Development Stages of Amylostereum areolatum

Frontiers in Microbiology ◽

10.3389/fmicb.2021.827241 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ningning Fu ◽

Jiaxing Li ◽

Ming Wang ◽

Lili Ren ◽

Shixiang Zong ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Growth And Development ◽

Target Genes ◽

Growth Stages ◽

Experimental Conditions ◽

Development Stages ◽

Symbiotic Fungus ◽

The Stability

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.

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

10.21203/rs.3.rs-35251/v3 ◽

2020 ◽

Author(s):

Huiyun Song ◽

Wenmai Mao ◽

Zhihao Duan ◽

Qingmin Que ◽

Wei Zhou ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Accurate Method ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Toona Ciliata

Abstract Background:Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem ( T. ciliata ). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.Results:The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta ( H. robusta ) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 ( TcMYB3) gene.Conclusions:This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

BioMed Research International ◽

10.1155/2015/823806 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Xiaoli Tang ◽

Hongyan Wang ◽

Chuyang Shao ◽

Hongbo Shao

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Profiles ◽

Elongation Factor ◽

Gene Expression Profiles ◽

Stable Reference Gene ◽

Experimental Conditions ◽

Suitable Reference

Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

Use of a lux reporter system for monitoring rapid changes in α-toxin gene expression in Clostridium perfringens during growth

FEMS Microbiology Letters ◽

10.1016/s0378-1097(00)00208-1 ◽

2000 ◽

Vol 188 (1) ◽

pp. 29-33 ◽

Cited By ~ 5

Author(s):

M Phillips-Jones

Keyword(s):

Gene Expression ◽

Clostridium Perfringens ◽

Toxin Gene ◽

Reporter System ◽

Rapid Changes ◽

Selection and Validation of Reference Genes for Gene Expression Analysis in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)

Insects ◽

10.3390/insects12070589 ◽

2021 ◽

Vol 12 (7) ◽

pp. 589

Author(s):

Xin Yan ◽

Yibo Zhang ◽

Kangkang Xu ◽

Yawei Wang ◽

Wenjia Yang

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Housekeeping Genes ◽

Tuta Absoluta ◽

Leaf Miner ◽

Specific Reference ◽

Internal Reference ◽

Experimental Conditions

The tomato leaf miner, Tuta absoluta is a destructive pest of tomato. The leaf-mining activities of its larvae can cause significant yield losses. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure gene expression, and the selection of stable reference genes for calibration and standardization is critical for accurate use of RT-qPCR. We studied the stable expression of nine common housekeeping genes in T. absoluta. These were examined at different developmental stages, in larval tissues, as well as those induced by exposure to 20E and insecticides. Four dedicated algorithms (geNorm, BestKeeper, NormFinder, and ΔCt method) and online tool (RefFinder) were used to analyze and rank the tested reference genes. Based on the standardized gene expression data of target gene ecdysone receptor (EcR), the applicability of specific reference genes was verified. The results clarify that the optimal internal reference genes vary greatly under different experimental conditions. GAPDH and RPS11 were the best reference genes for developmental stages; RPL28 and RPL10 for different tissues; EF1α and RPL28 for 20E treatment; EF1α and RPL7A for insecticide treatments. The most suitable reference genes in all experimental conditions are EF1α and RPL28.

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

BMC Plant Biology ◽

10.1186/s12870-020-02670-3 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Huiyun Song ◽

Wenmai Mao ◽

Zhihao Duan ◽

Qingmin Que ◽

Wei Zhou ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Accurate Method ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Toona Ciliata

Abstract Background Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. Results The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. Conclusions This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.