Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites

Abstract Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

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Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression inDioscorea opposita

BioMed Research International ◽

10.1155/2016/3089584 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13 ◽

Cited By ~ 7

Author(s):

Xiting Zhao ◽

Xiaoli Zhang ◽

Xiaobo Guo ◽

Shujie Li ◽

Linlin Han ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Chain Reaction ◽

Qrt Pcr ◽

Expression Studies ◽

Tuber Crop ◽

Polymerase Chain ◽

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 obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea oppositaThunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression ofPE2.1andPE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection inD. oppositaand will contribute toward more accurate gene analysis studies of the genusDioscorea.

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Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

Bulletin of Entomological Research ◽

10.1017/s000748531800072x ◽

2018 ◽

Vol 109 (4) ◽

pp. 443-452 ◽

Cited By ~ 1

Author(s):

C. Wang ◽

J. Yang ◽

Q. Pan ◽

S. Yu ◽

R. Luo ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Polymerase Chain Reaction ◽

Real Time Quantitative Pcr ◽

Expression Studies ◽

Gene Expression Studies ◽

The Stability ◽

Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

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Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-015-1884-8 ◽

2015 ◽

Vol 178 (3) ◽

pp. 433-450 ◽

Cited By ~ 18

Author(s):

Mogilicherla Kanakachari ◽

Amolkumar U. Solanke ◽

Narayanasamy Prabhakaran ◽

Israr Ahmad ◽

Gurusamy Dhandapani ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Solanum Melongena ◽

Expression Studies ◽

Gene Expression Studies ◽

Suitable Reference

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Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽

10.1139/gen-2017-0176 ◽

2018 ◽

Vol 61 (5) ◽

pp. 349-358 ◽

Cited By ~ 6

Author(s):

Yanchun You ◽

Miao Xie ◽

Liette Vasseur ◽

Minsheng You

Keyword(s):

Gene Expression ◽

Real Time ◽

Plutella Xylostella ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

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Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

Biologia Futura ◽

10.1556/019.70.2019.24 ◽

2019 ◽

Vol 70 (4) ◽

pp. 261-267

Author(s):

Gaigai Du ◽

Liyuan Wang ◽

Huawei Li ◽

Peng Sun ◽

Jianmin Fu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Developmental Stages ◽

Suitable Reference Gene ◽

Diospyros Kaki ◽

Stable Gene ◽

Expression Studies ◽

Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

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