Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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Selection of housekeeping genes for gene expression studies on the development of fruit bearing shoots in Chinese jujube (Ziziphus jujube Mill.)

Molecular Biology Reports ◽

10.1007/s11033-008-9433-y ◽

2008 ◽

Vol 36 (8) ◽

pp. 2183-2190 ◽

Cited By ~ 20

Author(s):

Hai-feng Sun ◽

Yu-ping Meng ◽

Gui-mei Cui ◽

Qiu-fen Cao ◽

Jie Li ◽

...

Keyword(s):

Gene Expression ◽

Housekeeping Genes ◽

Chinese Jujube ◽

Expression Studies ◽

Ziziphus Jujube ◽

Gene Expression Studies ◽

Selection Of

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Validation of housekeeping genes as an internal control for gene expression studies in the brain of ovariectomized rats treated with tibolone

Gene ◽

10.1016/j.gene.2020.145255 ◽

2021 ◽

Vol 769 ◽

pp. 145255

Author(s):

Iris A. Feria-Romero ◽

Iván Bribiesca-Cruz ◽

Angélica Coyoy-Salgado ◽

Julia J. Segura-Uribe ◽

Guadalupe Bautista-Poblet ◽

...

Keyword(s):

Gene Expression ◽

Internal Control ◽

Housekeeping Genes ◽

Ovariectomized Rats ◽

Expression Studies ◽

Gene Expression Studies ◽

The Brain

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Systematic identification of human housekeeping genes possibly useful as references in gene expression studies

Molecular Medicine Reports ◽

10.3892/mmr.2017.6944 ◽

2017 ◽

Vol 16 (3) ◽

pp. 2397-2410 ◽

Cited By ~ 24

Author(s):

Maria Caracausi ◽

Allison Piovesan ◽

Francesca Antonaros ◽

Pierluigi Strippoli ◽

Lorenza Vitale ◽

...

Keyword(s):

Gene Expression ◽

Housekeeping Genes ◽

Expression Studies ◽

Gene Expression Studies ◽

Systematic Identification

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Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes

10.7287/peerj.preprints.2125v1 ◽

2016 ◽

Author(s):

Joske Ruytinx ◽

Tony Remans ◽

Jan V Colpaert

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Intraspecific Variability ◽

Standard Technique ◽

Housekeeping Genes ◽

Ectomycorrhizal Fungus ◽

Suillus Luteus ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

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