Assessment of suitable reference genes for RT-qPCR studies in chronic rhinosinusitis.
Assessment of suitable reference genes for RT-qPCR studies in chronic rhinosinusitis.
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评估合适的参考基因用于慢性鼻炎的RT-QPCR研究。
DOI：
10.1038/s41598-018-19834-9
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发表时间：
2018-01-25
影响因子：
4.6
通讯作者：
Haruna SI
中科院分区：
文献类型：
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作者：
Nakayama T;Okada N;Yoshikawa M;Asaka D;Kuboki A;Kojima H;Tanaka Y;Haruna SI
逆转录-定量聚合酶链反应是一种有价值的、可靠的基因定量方法。靶基因表达通常通过使用参考基因（RG）的标准化来定量，并且准确的标准化对于产生可靠的数据至关重要。然而，在慢性鼻窦炎（CRS）患者的鼻息肉和鼻窦组织中稳定的RG尚未得到很好的研究。在这里，我们使用两阶段研究设计来识别稳定的RG。我们使用5个程序-geNorm、Normandy、BestKeeper、ΔCT和Refrigerant评估了15个常用候选RG的稳定性。核糖体蛋白侧柄亚基P1（RPLP 1）和核糖体蛋白侧柄亚基P0（RPLP 0）是第一阶段研究中最稳定的两个RG，这些结果在第二阶段得到验证。根据使用的所有算法，常用的RGs β-肌动蛋白（ACTB）和甘油醛3-磷酸脱氢酶（GAPDH）是不稳定的。通过使用稳定和不稳定的RG对IL-5、CCL 11、IFN-γ和IL-17 A进行相对定量，进一步验证了这些发现。根据所选RG的标准化，相对表达水平变化很大。适当选择稳定的RG将允许更准确地确定CRS患者中的靶基因表达水平。
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.
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影响因子：
12.4
作者：
Tokunaga T;Sakashita M;Haruna T;Asaka D;Takeno S;Ikeda H;Nakayama T;Seki N;Ito S;Murata J;Sakuma Y;Yoshida N;Terada T;Morikura I;Sakaida H;Kondo K;Teraguchi K;Okano M;Otori N;Yoshikawa M;Hirakawa K;Haruna S;Himi T;Ikeda K;Ishitoya J;Iino Y;Kawata R;Kawauchi H;Kobayashi M;Yamasoba T;Miwa T;Urashima M;Tamari M;Noguchi E;Ninomiya T;Imoto Y;Morikawa T;Tomita K;Takabayashi T;Fujieda S
通讯作者：
Fujieda S
影响因子：
--
作者：
Silver N;Best S;Jiang J;Thein SL
通讯作者：
Thein SL
影响因子：
6.4
作者：
Christensen, Jenna M.;Cheng, Jasmine;Harvey, Richard J.
通讯作者：
Harvey, Richard J.
影响因子：
14.9
作者：
Vermeulen J;De Preter K;Lefever S;Nuytens J;De Vloed F;Derveaux S;Hellemans J;Speleman F;Vandesompele J
通讯作者：
Vandesompele J
影响因子：
10
作者：
Glare, EM;Divjak, M;Walters, EH
通讯作者：
Walters, EH