Evaluation of Reference Genes for QPCR in Human Liver and Kidney Tissue from Individuals with Obesity

Overview

Journal Sci Rep

Specialty Science

Date 2025 Feb 13

PMID 39948154

Authors

Aleksandra Romaniuk-Drapala

Katarzyna Kosicka-Noworzyn

Yi-Hua Sheng

Christine Yohn

Luigi Brunetti

Leonid Kagan

Affiliations

Soon will be listed here.

Abstract

Given the obesity epidemic and the prevalence of comorbidities, there is an ongoing need to understand the health consequences of this disease state better. Understanding gene expression signals will facilitate the identification of mechanisms of kidney and liver dysfunction/disease often present in individuals with obesity. qPCR is the standard method for studying changes in relative gene expression. Reference genes (RGs) are obligatory for accurately normalizing mRNA transcript levels across samples. Despite the prevalence of qPCR, the reliability of the data is often compromised because RGs are still used without validation or have proven to be unstable in different tissues and various diseases. In this study, we validated seven reference genes (ACTB, B2M, RPLP0, HPRT1, GAPDH, 18S rRNA, and PPIA) using human liver tissue from 15 lean individuals and 17 individuals with a BMI ≥ 25 and human kidney tissue from 13 lean individuals and 15 individuals with a BMI ≥ 25. Cross-validation of expression stability was performed using the RefFinder platform with four algorithms: NormFinder, BestKeeper, geNorm, and the comparative ΔCt method. In obesity-related studies, the most suitable reference genes in gene expression studies are RPLP0 and HPRT1 in human kidney tissue and RPLP0 and GAPDH in the liver.

References

Huang T, Chen C, Du J, Zheng Z, Ye S, Fang S . A tRF-5a fragment that regulates radiation resistance of colorectal cancer cells by targeting MKNK1. J Cell Mol Med. 2023; 27(24):4021-4033. PMC: 10747413. DOI: 10.1111/jcmm.17982. View

Dheda K, Huggett J, Chang J, Kim L, Bustin S, Johnson M . The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem. 2005; 344(1):141-3. DOI: 10.1016/j.ab.2005.05.022. View

Fourdinier O, Glorieux G, Brigant B, Diouf M, Pletinck A, Vanholder R . Syndecan-1 and Free Indoxyl Sulfate Levels Are Associated with miR-126 in Chronic Kidney Disease. Int J Mol Sci. 2021; 22(19). PMC: 8508835. DOI: 10.3390/ijms221910549. View

Fan X, Yao H, Liu X, Shi Q, Lv L, Li P . High-Fat Diet Alters the Expression of Reference Genes in Male Mice. Front Nutr. 2020; 7:589771. PMC: 7732482. DOI: 10.3389/fnut.2020.589771. View

Mokdad A, Serdula M, Dietz W, Bowman B, Marks J, Koplan J . The spread of the obesity epidemic in the United States, 1991-1998. JAMA. 1999; 282(16):1519-22. DOI: 10.1001/jama.282.16.1519. View

Duan J, Nilsson L, Lambert B . Structural and functional analysis of mutations at the human hypoxanthine phosphoribosyl transferase (HPRT1) locus. Hum Mutat. 2004; 23(6):599-611. DOI: 10.1002/humu.20047. View

Meierhofer D, Weidner C, Sauer S . Integrative analysis of transcriptomics, proteomics, and metabolomics data of white adipose and liver tissue of high-fat diet and rosiglitazone-treated insulin-resistant mice identified pathway alterations and molecular hubs. J Proteome Res. 2014; 13(12):5592-602. DOI: 10.1021/pr5005828. View

Musso G, Gambino R, Tabibian J, Ekstedt M, Kechagias S, Hamaguchi M . Association of non-alcoholic fatty liver disease with chronic kidney disease: a systematic review and meta-analysis. PLoS Med. 2014; 11(7):e1001680. PMC: 4106719. DOI: 10.1371/journal.pmed.1001680. View

Shen H, Stoute J, Liu K . Structural and catalytic roles of the human 18 rRNA methyltransferases DIMT1 in ribosome assembly and translation. J Biol Chem. 2020; 295(34):12058-12070. PMC: 7443495. DOI: 10.1074/jbc.RA120.014236. View

10.

Marti A, Moreno-Aliaga M, Hebebrand J, Martinez J . Genes, lifestyles and obesity. Int J Obes Relat Metab Disord. 2004; 28 Suppl 3:S29-36. DOI: 10.1038/sj.ijo.0802808. View

11.

Silver N, Best S, Jiang J, Thein S . Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006; 7:33. PMC: 1609175. DOI: 10.1186/1471-2199-7-33. View

12.

Zhao J, Zhou L, Zhang Y, Cheng J, Zeng Y, Li X . TDF and TAF inhibit liver cancer cell migration, invasion via p7TP3. Sci Rep. 2024; 14(1):8161. PMC: 11001947. DOI: 10.1038/s41598-024-58807-z. View

13.

Taylor S, Mrkusich E . The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE). J Mol Microbiol Biotechnol. 2013; 24(1):46-52. DOI: 10.1159/000356189. View

14.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

15.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

16.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

17.

Wang J, Yu X, Cao X, Tan L, Jia B, Chen R . GAPDH: A common housekeeping gene with an oncogenic role in pan-cancer. Comput Struct Biotechnol J. 2023; 21:4056-4069. PMC: 10470192. DOI: 10.1016/j.csbj.2023.07.034. View

18.

Bangaru M, Park F, Hudmon A, McCallum J, Hogan Q . Quantification of gene expression after painful nerve injury: validation of optimal reference genes. J Mol Neurosci. 2011; 46(3):497-504. PMC: 3273664. DOI: 10.1007/s12031-011-9628-x. View

19.

Li T, Xu L, Wei Z, Zhang S, Liu X, Yang Y . ELF5 drives angiogenesis suppression though stabilizing WDTC1 in renal cell carcinoma. Mol Cancer. 2023; 22(1):184. PMC: 10656961. DOI: 10.1186/s12943-023-01871-2. View

20.

Ahrens M, Ammerpohl O, von Schonfels W, Kolarova J, Bens S, Itzel T . DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery. Cell Metab. 2013; 18(2):296-302. DOI: 10.1016/j.cmet.2013.07.004. View