Selection and Evaluation of Reference Genes for Quantitative Real-time PCR analysis in Lac Insect (Kerria lacca)
Selection and Evaluation of Reference Genes for Quantitative Real-time PCR analysis in Lac Insect (Kerria lacca)
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紫胶虫（Kerria lacca）实时定量 PCR 分析参考基因的选择和评估
DOI：
10.1111/1748-5967.12565
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发表时间：
2022
影响因子：
1.3
通讯作者：
Hang Chen
中科院分区：
文献类型：
--
作者：
Weiwei Wang;Xiaofei Ling;Nawaz Haider Bashir;Qin Lu;Jinwen Zhang;Tichu Li;Hang Chen
筛选和鉴定合适的内参基因是涉及定量实时聚合酶链式反应的实验的关键步骤。然而，对于具有重要经济意义的紫胶虫，紫胶虫的内参基因尚未进行研究。在本研究中，我们使用五种统计算法（ΔCt、geNorm、NormFinder、BestKeeper和RefFinder）评估八个候选内参基因（18S rRNA、28S rRNA、β-ACT、EF1-α、RPL10、RPL13、RPS11和RPS15）在K不同发育阶段和组织中的稳定性。 laccaand 接受 dsRNA 处理。我们的结果表明，发育阶段最稳定的内参基因是28S rRNA和RPS11，不同组织的内参基因是RPL13和RPS11，dsRNA处理的内参基因是β-ACT和EF1-α。虽然目标基因FAS和17β-HSD在标准化为稳定基因的表达水平时表现出一致的表达谱（单独或组合），但当使用最不稳定的参考基因（18S rRNA）时，这些表达谱发生了变化。这些结果强调应根据所使用的具体实验条件选择合适的内参基因。不同内参基因的可靠性和稳定性可以为紫胶昆虫基因表达分析提供坚实的基础。
The screening and identification of suitable reference genes are key steps in experiments involving the quantitative real‐time polymerase chain reaction. However, the reference genes ofKerria lacca, a lac insect with considerable economic significance, have not been investigated. In this study, we used five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of eight candidate reference genes (18S rRNA,28S rRNA,β‐ACT,EF1‐α,RPL10,RPL13,RPS11, andRPS15) in different developmental stages and tissues ofK. laccaand under dsRNA treatment. Our results showed that the most stable reference genes were28S rRNAandRPS11for the developmental stages,RPL13andRPS11for different tissues, andβ‐ACTandEF1‐αfor dsRNA treatments. Although the target genesFASand17β‐HSDshowed consistent expression profiles (alone or in combination) when normalized to the expression levels of the stable genes, those changed when the least stable reference gene (18S rRNA) was used. These results highlight that suitable reference genes should be chosen according to the specific experimental conditions used. The reliability and stability of different reference genes can provide a firm foundation for gene expression analysis in lac insects.