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

Abstract

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.

Selection and validation of suitable reference genes for gene expression studies in Callerya speciosa (Champ. ex Benth.) Schot under different experimental conditions

“…To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza 27 , Nitraria tangutorum 20 , Fucus distichus 28 , Bromus sterilis 19 , Allium tuberosum 29 , and Suaeda glauca 30 . In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth 31 , diabetic kidney disease 32 , Bronchopulmonary dysplasia 33 . Due to its rapidity and accuracy in informing on active coronavirus (CoV) infection, RT-qPCR has become the assay of choice for COVID-19 diagnosis 34,35 .…”

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

“…To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza, Nitraria tangutorum, Fucus distichus, Bromus sterilis, Allium tuberosum, and Suaeda glauca . In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth, diabetic kidney disease, and bronchopulmonary dysplasia . Due to its rapidity and accuracy in informing on active coronavirus ( CoV ) infection, RT-qPCR has become the assay of choice for COVID-19 diagnosis. , However, RT-qPCR requires a stable internal standard as a reference gene.…”

Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β

“…To define the best housekeeping gene and the best combination, we evaluate the cycle thresholds (Ct) value of RT-PCR in five different software applications: DataAssist (version 3.1; https://www.thermofsher.com/br/en/home/technical-resources/sofware-downloads/dataassist-sofware.html ), Bestkeeper (version 1.0; https://www.gene-quantifcation.de/bestkeeper.html ), RefFinder and the comparative ΔCt method ( https://www.heartcure.com.au/refnder/ ), GeNorm ( https://genorm.cmgg.be/ ), and NormFinder (version 0.953; https://moma.dk/normfnder-sofware ), following the authors’ recommendations. These software packages determine the relative expression stability of the candidate HKG and generate a rank of the best genes 16 . NormFinder is a freely available tool and recommends a standard deviation less than 0.5 (SD < 0.5).…”

“…Despite the growing number of studies investigating the reference genes for renal disease models 10 , 16 , 17 , to our knowledge there are no detailed reports selecting the most stable gene recommended for the frequently used in vitro model of TGF-β-induced fibrogenesis in MCs. This limitation may lead to non-reproducible data.…”