Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

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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Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

BioMed Research International ◽

10.1155/2015/363575 ◽

2015 ◽

Vol 2015 ◽

pp. 1-20 ◽

Cited By ~ 6

Author(s):

Cora S. Thiel ◽

Swantje Hauschild ◽

Svantje Tauber ◽

Katrin Paulsen ◽

Christiane Raig ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Housekeeping Genes ◽

Altered Gravity ◽

Experimental Conditions ◽

Expression Levels ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

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Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽

10.3390/plants10071272 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1272

Author(s):

Judit Tajti ◽

Magda Pál ◽

Tibor Janda

Keyword(s):

Gene Expression ◽

Avena Sativa ◽

Abiotic Stresses ◽

Reference Genes ◽

Nutritional Value ◽

Tissue Type ◽

Future Research ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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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 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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