Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

Download Full-text

Identification of suitable reference genes for gene expression analysis of pork meat quality and analysis of candidate genes associated with the trait drip loss

Meat Science ◽

10.1016/j.meatsci.2010.05.030 ◽

2010 ◽

Vol 86 (2) ◽

pp. 436-439 ◽

Cited By ~ 17

Author(s):

Jean McBryan ◽

Ruth M. Hamill ◽

Grace Davey ◽

Peadar Lawlor ◽

Anne Maria Mullen

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Meat Quality ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Drip Loss ◽

Pork Meat ◽

Suitable Reference

Download Full-text

Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR

PLoS ONE ◽

10.1371/journal.pone.0220475 ◽

2019 ◽

Vol 14 (8) ◽

pp. e0220475 ◽

Cited By ~ 4

Author(s):

Lingling Zhang ◽

Xiaodong Jing ◽

Wei Chen ◽

Jianlin Bai ◽

Liette Vasseur ◽

...

Keyword(s):

Expression Analysis ◽

Plutella Xylostella ◽

Reference Genes ◽

Qrt Pcr ◽

Selection Of

Download Full-text

Selection and validation of reference genes for normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

10.21203/rs.3.rs-394417/v1 ◽

2021 ◽

Author(s):

Michihito Deguchi ◽

Shobha Potlakayala ◽

Zachary Spuhler ◽

Hannah George ◽

Vijay Sheri ◽

...

Keyword(s):

Gene Expression ◽

Heavy Metal ◽

Expression Analysis ◽

Reference Genes ◽

Cannabis Sativa ◽

Expression Profiles ◽

Expression Patterns ◽

Qrt Pcr ◽

Differential Gene ◽

Industrial Hemp

Abstract Industrial hemp (Cannabis sativa L.) is a dioecious crop widely known for its production of phytocannabinoids, flavonoids, and terpenes. In the past two years since its legalization, there has been significant interest in researching this important crop for pharmaceutical applications. Although many scientific reports have demonstrated gene expression analysis of hemp through OMICs approaches, accurate validation of omics data cannot be performed because of lack of reliable reference genes for normalization of qRT-PCR data. The differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stress were evaluated through four software packages: geNorm, NormFinder, BestKeeper, and RefFinder. The EF-1a ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal and hormonal stress. The expression profiles of two cannabinoid pathway genes, AAE1 and THCAS, using the two most stable and single least stable reference genes confirmed that two most stables genes were apt for normalization of gene expression data. This work will contribute to the future studies on the expression analysis of hemp genes regulating the synthesis, transport and accumulation of secondary metabolites.

Download Full-text

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

10.1101/2020.08.12.247601 ◽

2020 ◽

Author(s):

Liz M. Florez ◽

Reiny W. A. Scheper ◽

Brent M. Fisher ◽

Paul W. Sutherland ◽

Matthew D. Templeton ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Time Course ◽

Gene Expression Analysis ◽

Virulence Genes ◽

Functional Characterization ◽

Post Inoculation ◽

In Planta ◽

Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

Download Full-text

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.

Download Full-text

Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots

PLoS ONE ◽

10.1371/journal.pone.0226168 ◽

2019 ◽

Vol 14 (12) ◽

pp. e0226168 ◽

Cited By ~ 2

Author(s):

Li Li ◽

Kangyu Wang ◽

Mingzhu Zhao ◽

Shaokun Li ◽

Yue Jiang ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Hairy Roots ◽

Reference Genes ◽

Gene Expression Analysis ◽

Qrt Pcr

Download Full-text

Determination of Suitable Candidate Genes as Endogenous Control for Gene Expression Analysis in Local Capsicum Annuum MC11

Turkish Journal of Computer and Mathematics Education (TURCOMAT) ◽

10.17762/turcomat.v12i3.837 ◽

2021 ◽

Vol 12 (3) ◽

pp. 1011-1017

Author(s):

Marina Mokhtar Et.al

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Capsicum Annuum ◽

Reference Genes ◽

Data Normalization ◽

Plant Parts ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies ◽

Suitable Reference

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for qRT-PCR assays. This study identifies suitable reference genes for local chilli, Capsicum annuum var MC11 under incident of Cucumber mosaic virus infection. Six candidate genes actin, tub, EF1α, GAPDH, TEF1α and 18SrRNA and three validated Capsicum reference genes UBI-3 ref, β-tub ref and gapdhref were tested against five chilli plant parts stem, shoot, leave, flower and root. The PCR/qRT-PCR results demonstrate only five candidate references genes actin, EF1α, GAPDH, 18SrRNA, and TEF1α that show specific single band of amplicon, without primer dimers and at the targeted sizes. Through qRT-PCR, GAPDH gives single peak in dissociation curve in all plant parts used further fulfilling the characteristic of reference genes.Previous work on validation of reference genes in pepper shows that only UBI-3 suits to C. annuum var MC11 infected CMV, thus we suggest that GAPDH has a potential to be a validated reference gene for C. annuum var MC11 and can be used together UBI-3 for the purpose of data normalization.

Download Full-text