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.
Highlights
Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) is routinely used for gene expression analysis
In this study we examined the expression levels of the 15 reference genes (RGs) provided in the Human Housekeeping Gene Primer Set (Takara, Shiga, Japan) (Table 1) in 39 patients in the first stage of the study, and in 36 patients in the second stage
The PCR efficiency of each RG is shown in Supplementary Table S1
Summary
Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) is routinely used for gene expression analysis. The pathophysiology of CRS has not been well elucidated, numerous studies have been published to date that have revealed some of the underlying mechanism. In these previous studies of CRS, traditional RGs such as β-actin (ACTB), glyceraldehyde-3-phosphate hydrogenase (GAPDH), and β-glucuronidase (GUSB) were used for RT–qPCR normalization[11,12,13,14]. We applied a two-stage study design and investigated the stability of potential RGs in nasal polyps and sinonasal tissues to determine appropriate RGs for analysing target gene expression levels in CRS. 194 138 75 131 94 200 108 166 92 89 170 194 interleukin (IL)-5, interferon (IFN)-γ, and IL-17A, which are the major Th1/Th2/Th17 cell cytokines, and CCL11 which is the eosinophil chemoattractant chemokine were evaluated as target genes using the most stable RGs and conventional, less stable RGs for normalization
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