Identification of suitable reference genes for RT-qPCR studies in human parathyroid tissue glandular cells.

Abstract

Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. The quantitative method of gene expression analyses via qRT-PCR provides an accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue and glandular cells obtained from primary and secondary hyperparathyroidism patients were isolated enzymatically by removing extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found that RPLP0>GAPDH>PGK1. Lastly, relative target gene (GCM2) expression comparisons revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue