Selection and validation of reference genes for qRT-PCR analysis of gene expression in Microsporum canis growing under different adhesion-inducing conditions

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

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Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

PLoS ONE ◽

10.1371/journal.pone.0169465 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169465 ◽

Cited By ~ 6

Author(s):

Dongli Wan ◽

Yongqing Wan ◽

Qi Yang ◽

Bo Zou ◽

Weibo Ren ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Environmental Stresses ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

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Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Chinese Journal of Oceanology and Limnology ◽

10.1007/s00343-015-4004-2 ◽

2014 ◽

Vol 32 (6) ◽

pp. 1248-1256 ◽

Cited By ~ 9

Author(s):

Ye Zhao ◽

Muyan Chen ◽

Tianming Wang ◽

Lina Sun ◽

Dongxue Xu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Sea Cucumber ◽

Apostichopus Japonicus ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

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Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

PeerJ ◽

10.7717/peerj.7133 ◽

2019 ◽

Vol 7 ◽

pp. e7133 ◽

Cited By ~ 4

Author(s):

Wen Zhou ◽

Shiqiang Wang ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Pcr Analysis ◽

Potential Candidate ◽

Qrt Pcr ◽

Expression Studies ◽

Different Tissues

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.

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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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Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury)

Insects ◽

10.3390/insects13010097 ◽

2022 ◽

Vol 13 (1) ◽

pp. 97

Author(s):

Xudong Zhao ◽

Yishu Geng ◽

Tianyi Hu ◽

Yongang Zhao ◽

Suling Yang ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Comprehensive Evaluation ◽

Larval Density ◽

Physiological Role ◽

Invasive Pest ◽

Pcr Analysis ◽

Hyphantria Cunea ◽

Experimental Conditions ◽

Qrt Pcr

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.

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Selection and Evaluation of Potential Reference Genes for Quantitative Real-Time PCR in Agaricus blazei Based on Transcriptome Sequencing Data

BioMed Research International ◽

10.1155/2021/6661842 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Yuan-Ping Lu ◽

Jian-Hua Liao ◽

Zhong-Jie Guo ◽

Hui-Qing Zheng ◽

Ling-Fang Lu ◽

...

Keyword(s):

Gene Expression ◽

Cytochrome C ◽

Expression Pattern ◽

Real Time Pcr ◽

Reference Genes ◽

Fruit Body ◽

Pcr Analysis ◽

Agaricus Blazei ◽

Quantitative Real Time Pcr ◽

Qrt Pcr

Quantitative real-time PCR (qRT-PCR) is widely used to detect gene expression due to its high sensitivity, high throughput, and convenience. The accurate choice of reference genes is required for normalization of gene expression in qRT-PCR analysis. In order to identify the optimal candidates for gene expression analysis using qRT-PCR in Agaricus blazei, we studied the potential reference genes in this economically important edible fungus. In this study, transcriptome datasets were used as source for identification of candidate reference genes. And 27 potential reference genes including 21 newly stable genes, three classical housekeeping genes, and homologous genes of three ideal reference genes in Volvariella volvacea, were screened based on transcriptome datasets of A. blazei and previous studies. The expression stability of these genes was investigated by qRT-PCR analysis and further evaluated by four software packages, geNorm, NormFinder, BestKeeper, and RefFinder. Among these candidates, α-TUB (Tubulin alpha) and Cox5a (COX5A subunit VA of cytochrome c oxidase) were revealed as the most stable in fruit body, and suitable for 5 different developmental stages. α-TUB and ATP3 (ATP3 gamma subunit of the F1 sector of mitochondrial F1F0 ATP synthase) showed the most stable expression in stipe tissues and, Uqcrc (core subunit of the ubiquinol-cytochrome c reductase complex) and PUP3 (20S proteasome subunit beta 3) performed well in pileus tissues during the process of A. blazei development, while GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was among the least stable genes in all sample sets. Finally, the Ableln3 (homology of eln3 gene of Coprinus cinereus) was adopted to validate the reliability of these stable and unstable reference genes, indicating that the use of unsuitable reference genes as internal controls could change the target gene’s expression pattern. This study can provide guidance for choosing reference genes for analyzing the expression pattern of target genes and facilitate the functional genomic investigation on fruit body formation and development, as well as stipe elongation and pileus expansion in A. blazei.

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Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling

Scientific Reports ◽

10.1038/s41598-021-95849-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jing Tong ◽

Manman Hu ◽

Beibei Han ◽

Yanhai Ji ◽

Baoju Wang ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Transcriptome Profiling ◽

Pcr Analysis ◽

Allium Tuberosum ◽

Qrt Pcr ◽

Complex Processes ◽

Chinese Chive ◽

Gene Expression Studies ◽

The Stability

AbstractChinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

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Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

Biomarker Insights ◽

10.4137/bmi.s5596 ◽

2010 ◽

Vol 5 ◽

pp. BMI.S5596 ◽

Cited By ~ 7

Author(s):

Yi-Hong Zhou ◽

Vinay R. Raj ◽

Eric Siegel ◽

Liping Yu

Keyword(s):

Gene Expression ◽

Real Time ◽

Gene Expression Data ◽

Reference Genes ◽

Pcr Analysis ◽

Expression Data ◽

Internal Reference ◽

Expression Ratio ◽

Qrt Pcr ◽

Single Standard

In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients–-a process that requires large sample sizes by combining independent sets of data.

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Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

International Journal of Molecular Sciences ◽

10.3390/ijms22052569 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2569

Author(s):

Xue Bai ◽

Tao Chen ◽

Yuan Wu ◽

Mingyong Tang ◽

Zeng-Fu Xu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Expression Patterns ◽

Pcr Analysis ◽

Cyperus Esculentus ◽

Transcriptome Data ◽

Qrt Pcr ◽

Below Ground ◽

The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

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