Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae)
Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae),...
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MDPI AG
2023-12-01
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| Series: | Insects |
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| Online Access: | https://www.mdpi.com/2075-4450/14/12/960 |
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| author | Liuyang Wang Qingyu Liu Pei Guo Zhanlin Gao Dan Chen Tao Zhang Jun Ning |
| author_facet | Liuyang Wang Qingyu Liu Pei Guo Zhanlin Gao Dan Chen Tao Zhang Jun Ning |
| author_sort | Liuyang Wang |
| collection | DOAJ |
| description | Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (<i>RPL32</i>, <i>RPS23</i>, <i>SDHA</i>, <i>UBQ</i>, <i>UCCR</i>, <i>GST</i>, <i>TATA</i>−<i>box</i>, <i>HSP70</i>, <i>GAPDH</i>, <i>RPL7A</i>, <i>SOD</i>, <i>RPS3</i>, <i>Actin</i>, α−<i>tubulin</i>, <i>AK</i>, and <i>EF1</i>) were carefully chosen in <i>R. pedestris</i>, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in <i>R. pedestris</i> is contingent upon the specific experimental conditions. <i>RPL7A</i> and <i>EF1</i> are optimal reference genes for developmental stages. Furthermore, α−<i>tubulin</i> and <i>EF1</i> exhibit the most stable expression across various adult tissues. <i>RPL32</i> and <i>RPL7A</i> exhibit the most stable expression for adult age. For nymph age, <i>RPL32</i> and <i>SOD</i> display the most stable expression. For temperature conditions, <i>RPS23</i> and <i>RPL7A</i> were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (<i>RpedOBP37</i>) and cytochrome P450 6a2 (<i>RpedCYP6</i>) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on <i>R. pedestris</i>, serving as a fundamental groundwork for future investigations on stable reference genes in <i>R. pedestris</i> as well as other organisms. |
| first_indexed | 2024-03-08T20:39:53Z |
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| language | English |
| last_indexed | 2024-03-08T20:39:53Z |
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| spelling | doaj.art-769432ca17074f268a4cbc2a86103d622023-12-22T14:16:21ZengMDPI AGInsects2075-44502023-12-01141296010.3390/insects14120960Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae)Liuyang Wang0Qingyu Liu1Pei Guo2Zhanlin Gao3Dan Chen4Tao Zhang5Jun Ning6State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture, Baoding 071000, ChinaInstitute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture, Baoding 071000, ChinaInstitute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture, Baoding 071000, ChinaInstitute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture, Baoding 071000, ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaQuantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (<i>RPL32</i>, <i>RPS23</i>, <i>SDHA</i>, <i>UBQ</i>, <i>UCCR</i>, <i>GST</i>, <i>TATA</i>−<i>box</i>, <i>HSP70</i>, <i>GAPDH</i>, <i>RPL7A</i>, <i>SOD</i>, <i>RPS3</i>, <i>Actin</i>, α−<i>tubulin</i>, <i>AK</i>, and <i>EF1</i>) were carefully chosen in <i>R. pedestris</i>, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in <i>R. pedestris</i> is contingent upon the specific experimental conditions. <i>RPL7A</i> and <i>EF1</i> are optimal reference genes for developmental stages. Furthermore, α−<i>tubulin</i> and <i>EF1</i> exhibit the most stable expression across various adult tissues. <i>RPL32</i> and <i>RPL7A</i> exhibit the most stable expression for adult age. For nymph age, <i>RPL32</i> and <i>SOD</i> display the most stable expression. For temperature conditions, <i>RPS23</i> and <i>RPL7A</i> were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (<i>RpedOBP37</i>) and cytochrome P450 6a2 (<i>RpedCYP6</i>) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on <i>R. pedestris</i>, serving as a fundamental groundwork for future investigations on stable reference genes in <i>R. pedestris</i> as well as other organisms.https://www.mdpi.com/2075-4450/14/12/960<i>Riptortus pedestris</i>qRT-PCRreference genesexpression stabilitynormalization |
| spellingShingle | Liuyang Wang Qingyu Liu Pei Guo Zhanlin Gao Dan Chen Tao Zhang Jun Ning Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) Insects <i>Riptortus pedestris</i> qRT-PCR reference genes expression stability normalization |
| title | Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) |
| title_full | Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) |
| title_fullStr | Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) |
| title_full_unstemmed | Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) |
| title_short | Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, <i>Riptortus pedestris</i> (Hemiptera: Alydidae) |
| title_sort | evaluation of reference genes for quantitative real time pcr analysis in the bean bug i riptortus pedestris i hemiptera alydidae |
| topic | <i>Riptortus pedestris</i> qRT-PCR reference genes expression stability normalization |
| url | https://www.mdpi.com/2075-4450/14/12/960 |
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