Validation and application of normalization factors for gene expression studies in rubella virus‐infected cell lines with quantitative real‐time PCR

Abstract

Reference genes are generally employed in real-time quantitative PCR (RT-qPCR) experiments to normalize variability between different samples. The aim of this study was to identify and validate appropriate reference genes as internal controls for RT-qPCR experiments in rubella virus (RV)-infected Vero and MCF-7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the DeltaDeltaC(t) calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT-qPCR analysis of RV-infected Vero and MCF-7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin-dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV-infected cells. The effect on cellular gene transcription by RV-infection seems to be cell line-specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV-infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT-qPCR data and the host-cell response to RV-infection are strictly cell-line specific.

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

“…These differences resulted from the heterogeneous gene expression profile of the tumor type, the experimental conditions, and the stability of the reference genes, being in agreement with the literature (Supplementary Table 1) [21,22,28,29]. In addition, the aforementioned variability can also be attributed to the different algorithms applied by the GeNorm and NormFinder [23,[30][31][32][33]. However, for the single HKG analysis, the first three ranked genes by GeNorm and NormFinder were the same ones, implying no substantial differences in their stability.…”

Quantitative Proteomic Analysis Reveals Antiviral and Anti-inflammatory Effects of Puerarin in Piglets Infected With Porcine Epidemic Diarrhea Virus

“…The relative expression level of each gene was calculated with the 2 − CT method (29). Monkey hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as the reference gene in Vero cells (30). Porcine ribosomal protein L4 (RPL4) was used as the reference gene in the jejunum, ileum, and mesenteric lymph nodes (28).…”

A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease

“…First-strand cDNA was generated as described above, and real-time PCR was performed assessing for genome RNA using the primers 5′-AGCCAACCAACCTCGATCT CTTGT-3′ (forward) and 5′-TGACACCAAGAACAAGGCTCTCCA-3′ (reverse). cDNA was normalized using the GAPDH primers 5′-TGCACCACCAA CTGCTTAGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse) 42 . Normalized results were then compared as ratios of MA-ExoN to MAwt genomes using the ∆∆Ct method.…”