Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

ABSTRACTBackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high quality qPCR, providing strong support for results, as well as performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used so far to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g. Losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cells and podocytes studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n=5; 5mM glucose), mannitol (n=5; 30mM, as osmotic control), HG (n=5; 30mM glucose), and HG + losartan (n=5; 30mM glucose and 10-4 mM of losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes is the best combination for qPCR normalization for both mesangial cell and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR technique, while for podocytes, HPRT and GAPDH should be chosen.

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Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

PLoS ONE ◽

10.1371/journal.pone.0246227 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0246227

Author(s):

Nicole Dittrich Hosni ◽

Ana Carolina Anauate ◽

Mirian Aparecida Boim

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

High Glucose ◽

Real Time Pcr ◽

Reference Genes ◽

Mesangial Cell ◽

Mesangial Cells ◽

Expression Studies ◽

Gene Expression Studies

Background Real-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high-quality qPCR, providing strong support for results, and performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used thus far to enhance the validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g., losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cell and podocyte studies of an in vitro DKD model of disease and its treatment. Methods Five software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted, and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n = 5; 5 mM glucose), mannitol (n = 5; 30 mM, as osmotic control), HG (n = 5; 30 mM glucose), and HG + losartan (n = 5; 30 mM glucose and 10−4 mM losartan). Real-time PCR was performed according to MIQE guidelines. Results We identified that the use of 2 genes was the best combination for qPCR normalization for both mesangial cells and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results. Conclusion This analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR, while for podocytes, HPRT and GAPDH should be chosen.

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Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PLoS ONE ◽

10.1371/journal.pone.0046451 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46451 ◽

Cited By ~ 176

Author(s):

Deshui Liu ◽

Lindan Shi ◽

Chenggui Han ◽

Jialin Yu ◽

Dawei Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Nicotiana Benthamiana ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

Scientific Reports ◽

10.1038/s41598-018-27420-2 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 16

Author(s):

Ana Érika Inácio Gomes ◽

Leonardo Prado Stuchi ◽

Nathália Maria Gonçalves Siqueira ◽

João Batista Henrique ◽

Renato Vicentini ◽

...

Keyword(s):

Gene Expression ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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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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