Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, 710061, China.
Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, 710061, China.
Exp Parasitol. 2019 Nov;206:107754. doi: 10.1016/j.exppara.2019.107754. Epub 2019 Aug 29.
Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.
粉尘螨是室内过敏原的重要来源，对环境温度具有很强的耐受性。然而，其耐受的规律和机制尚不清楚。基于我们之前对温度胁迫下粉尘螨的 RNA-seq 分析和注释，计划通过定量实时 PCR（qRT-PCR）鉴定参与温度胁迫反应的差异表达基因（DEGs）。然而，缺乏参考基因直接限制了 DEGs 的检测和确认。因此，在本研究中，我们选择了 6 个候选基因作为粉尘螨的参考基因：60S RP L11、60S RP L21、α-微管蛋白、GAPDH、Der f Mal f 6 和钙网蛋白，并使用 geNorm、NormFinder、BestKeeper、比较 ΔCt 和 RefFinder 方法评估它们在受热和受冷胁迫时的表达稳定性。然后检测和验证了 15 个 DEGs 的表达水平。geNorm 分析表明，α-微管蛋白和钙网蛋白是粉尘螨受热和受冷胁迫时最稳定的参考基因。NormFinder 和 BestKeeper 也得到了类似的评价结果，其中 60S RP L21 和 α-微管蛋白是最稳定的参考基因。通过比较 ΔCt 法和 RefFinder 的综合评价，确定 α-微管蛋白是粉尘螨受热和受冷胁迫时最理想的参考基因。此外，15 个 DEGs 的 qRT-PCR 检测结果与 RNA-seq 结果几乎一致，表明α-微管蛋白作为参考基因稳定。本研究为 qRT-PCR 检测粉尘螨 DEGs 表达提供了技术支持。
