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

Author(s)

dos Santos Bronel, Bruno Aristides ; Anauate, Ana Carolina ; Maquigussa, Edgar ; Boim, Mirian Aparecida ; Novaes, Antonio da Silva

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 12, n. 1, p. 10-pg., 2022-09-17.

Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-beta. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and alpha-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and alpha-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-beta. (AU)

• FAPESP's process: 15/23345-9 - MicroRNAs, extracellular vesicles and stem cells: physiology, pathophysiological role and therapeutic potential in renal diseases
• Grantee: Mirian Aparecida Boim
• Support Opportunities: Research Projects - Thematic Grants