Abstract: TH-PO889
Selection of Suitable Housekeeping Genes for Mesangial Cell Studies with High Glucose and Angiotensin II Receptor Blocker
Session Information
- Diabetic Kidney Disease: Basic - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Authors
- Hosni, Nicole Dittrich, Universidade Federal de São Paulo, São Paulo, Brazil
- Anauate, Ana Carolina, Universidade Federal de São Paulo, São Paulo, Brazil
- Boim, Mirian A., Universidade Federal de São Paulo, São Paulo, Brazil
Background
Real-time PCR (qPCR) is currently the gold standard method to gene expression studies. Identification of the best reference gene is a key point to perform high quality qPCR, being able to provide strong support for results, as well as acting as a source of bias when inappropriately chosen. Mesangial cells (MC), as an essential cell line in diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the most excellent housekeeping (HK) gene to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, being angiotensin II receptor blockers (ex. Losartan) the most commonly used. Our objective was to evaluate the suitability and define the most stable reference gene for MC 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 adopted to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PP1A, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse MC 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). qPCR was performed according to MIQE guidelines in QuantStudio 7Flex (Applied Biosystems).
Results
HPRT presented higher stability values in RefFinder, ΔCt method, and NormFinder softwares (Table 1), while frequently used HK such as GAPDH and ACTB (Biederman et al. 2004) showed lower scores compared to HPRT.
Conclusion
This analysis provides support to the use of HPRT as a HK gene in mouse mesangial cell studies of gene expression via qPCR technique.
Ranking of the candidate reference genes by each method used. Lower values indicate higher stability in gene expression.
| RefFinder | Geomean | ΔCt method | Mean SD | NormFinder* | Stability value | GeNorm | M value | BestKeeper | CV | SD | DataAssist | Score |
| HPRT | 1.00 | HPRT | 0.67 | HPRT | 0.118 | ACTB | 0.031 | ACTB | 2.96 | 0.87 | PGAM-1 | 5.324 |
| ACTB | 2.21 | ACTB | 0.75 | ACTB | 0.158 | GAPDH | 0.031 | HPRT | 2.99 | 0.70 | PP1A | 5.514 |
| PP1A | 3.41 | PGAM-1 | 0.77 | GAPDH | 0.179 | PGAM-1 | 0.0341 | B2M | 3.03 | 0.57 | HPRT | 5.525 |
| PGAM-1 | 3.94 | PP1A | 0.77 | PGAM-1 | 0.181 | HPRT | 0.0380 | PP1A | 3.41 | 0.63 | ACTB | 5.624 |
| GAPDH | 4.47 | GAPDH | 0.86 | PP1A | 0.226 | PP1A | 0.0390 | GAPDH | 3.51 | 0.79 | GAPDH | 5.907 |
| B2M | 4.56 | B2M | 0.92 | B2M | 0.244 | B2M | 0.0422 | PGAM-1 | 3.65 | 0.80 | B2M | 6.835 |
SD, standard deviation; CV, coefficient of variation. *Best reference genes analyzed by NormFinder considering the intra- and intergroup variations.
Funding
- Government Support - Non-U.S.