Validation of the Reference Genes for the Gene Expression Studies in Different Cell Lines of Pig

Abstract

Reverse transcription quantitative real-time polymerase chain reaction is one of the important methods to investigate gene expression in cells and tissues. However, if the data cannot be normalized with appropriate reference genes, the results may be unreliable. In this study, we detected the expression of 15 reference genes in three pig cell lines. The results showed that SDHA and ALDOA were the most stable reference genes in 3D4/21 cells. TOP2B, TBP, and PPIA were the most stable reference genes in PK-15 cells. SDHA and ALDOA were the most stable reference genes in IPEC-J2 cells. In addition, each cell line only needs to use two reference genes to standardize the expression of target genes. Taken together, this study provides a reference for different pig cell lines to select reference genes and also provides a theoretical basis for the use of these cell lines in related functional researches.

Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

“…It is a primary cell that easily adapts its gene expression which has been studied in both in vivo and in vitro studies 22 . Up till now, control genes were selected for studies on the role of cell and tissue type and the effect of bacterial pathogenic molecules, such as LPS and LTA in pigs 15 , 21 . However, reference genes in PAMs to follow the effects of a viral infection were not evaluated and validated yet.…”

“…However, expression levels of reference genes among the biological samples fluctuated in PAMs after cultivation or viral infection. In pigs, a number of research papers have been published in recent years examining endogenous control genes for normalization of gene expression data in different cell types, various tissues, and cell lines 15 , 21 , 23 – 28 . Gu et al 29 performed an extensive study for the validation of 20 endogenous control genes in 56 tissue types in pigs.…”

“…Multiple algorithms have been developed to evaluate reference genes 16 – 20 . In pigs, reference genes have been evaluated in different cell lines 21 and alveolar macrophages upon exposure to bacterial pathogenic molecules 15 . However, reference genes have never been validated in gene expression studies of PAMs during in vitro viral infections.…”

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

“…The algorithms used in this study, geNorm 59 , NormFinder 62 , BestKeeper 63 , Delta-Ct 64 , and RefFinder 65,66 have been commonly used to evaluate the stability of reference genes in plants 19,25,54 , animals [67][68][69] and microorganisms [70][71][72] . They are used to calculate the stability of gene expression using different mathematical models.…”