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, 8 (1), 1568

Assessment of Suitable Reference Genes for RT-qPCR Studies in Chronic Rhinosinusitis

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Assessment of Suitable Reference Genes for RT-qPCR Studies in Chronic Rhinosinusitis

Tsuguhisa Nakayama et al. Sci Rep.

Abstract

Reverse transcription-quantitative polymerase chain reaction is a valuable and reliable method for gene quantification. Target gene expression is usually quantified by normalization using reference genes (RGs), and accurate normalization is critical for producing reliable data. However, stable RGs in nasal polyps and sinonasal tissues from patients with chronic rhinosinusitis (CRS) have not been well investigated. Here, we used a two-stage study design to identify stable RGs. We assessed the stability of 15 commonly used candidate RGs using five programs-geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. Ribosomal protein lateral stalk subunit P1 (RPLP1) and ribosomal protein lateral stalk subunit P0 (RPLP0) were the two most stable RGs in the first stage of the study, and these results were validated in the second stage. The commonly used RGs β-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unstable according to all of the algorithms used. The findings were further validated via relative quantification of IL-5, CCL11, IFN-γ, and IL-17A using the stable and unstable RGs. The relative expression levels varied greatly according to normalization with the selected RGs. Appropriate selection of stable RGs will allow more accurate determination of target gene expression levels in patients with CRS.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Stability ranking of candidate reference genes by geNorm in the first stage of the study. (A) Gene expression stability (geNorm M) in all samples. Least stable to the left and most stable to the right. (B) Determination of the optimal number of reference genes. The V2/3 value was below the 0.15 threshold and the optimal number of reference genes was two. (C) Gene expression stability (geNorm M) in subgroups.
Figure 2
Figure 2
Stability ranking of candidate reference genes by geNorm in the second stage of the study. (A) Gene expression stability (geNorm M) in all samples. (B) Determination of the optimal number of reference genes. The V2/3 value was below the 0.15 threshold. (C) Gene expression stability (geNorm M) in subgroups.
Figure 3
Figure 3
Effect of reference gene selection on relative quantification of IL-5, CCL11, IFN-γ, and IL-17A mRNA expression. Error bars represent standard deviation. Kruskal–Wallis with post-hoc Dunn’s multiple comparison tests were used. *p < 0.05, **p < 0.01.

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