Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae)
Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (q...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-12-01
|
| Series: | Genes |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4425/12/1/21 |
| _version_ | 1797543485693231104 |
|---|---|
| author | Yu Bai Ya-Nan Lv Mei Zeng Pei-Yao Jia Hu-Na Lu Yi-Bo Zhu Sheng Li Ying-Ying Cui Yun-Xia Luan |
| author_facet | Yu Bai Ya-Nan Lv Mei Zeng Pei-Yao Jia Hu-Na Lu Yi-Bo Zhu Sheng Li Ying-Ying Cui Yun-Xia Luan |
| author_sort | Yu Bai |
| collection | DOAJ |
| description | Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from <i>Thermobia domestica</i>, a representative species of Zygentoma, including <i>Actin 5C</i> (<i>Actin5C</i>), <i>Elongation factor-1 alpha</i> (<i>EF1A</i>), <i>Ribosome protein S26</i> (<i>RPS26</i>), <i>Ribosome protein L32</i> (<i>RPL32</i>), and <i>Superoxide dismutase 2</i> (<i>SOD2</i>), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of <i>RPS26</i> and <i>RPL32</i> was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of <i>Myoglianin</i> (<i>Myo</i>), a crucial gene-regulating insect metamorphosis, in ametabolous <i>T. domestica</i>, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma. |
| first_indexed | 2024-03-10T13:46:16Z |
| format | Article |
| id | doaj.art-2f72f21162fd4c2db67bbef279138529 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-10T13:46:16Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-2f72f21162fd4c2db67bbef2791385292023-11-21T02:34:42ZengMDPI AGGenes2073-44252020-12-011212110.3390/genes12010021Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae)Yu Bai0Ya-Nan Lv1Mei Zeng2Pei-Yao Jia3Hu-Na Lu4Yi-Bo Zhu5Sheng Li6Ying-Ying Cui7Yun-Xia Luan8Guangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaGuangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, ChinaZygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from <i>Thermobia domestica</i>, a representative species of Zygentoma, including <i>Actin 5C</i> (<i>Actin5C</i>), <i>Elongation factor-1 alpha</i> (<i>EF1A</i>), <i>Ribosome protein S26</i> (<i>RPS26</i>), <i>Ribosome protein L32</i> (<i>RPL32</i>), and <i>Superoxide dismutase 2</i> (<i>SOD2</i>), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of <i>RPS26</i> and <i>RPL32</i> was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of <i>Myoglianin</i> (<i>Myo</i>), a crucial gene-regulating insect metamorphosis, in ametabolous <i>T. domestica</i>, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.https://www.mdpi.com/2073-4425/12/1/21<i>Thermobia domestica</i>reference genesexpression stabilityquantitative real-time PCRRNA <i>interference</i> |
| spellingShingle | Yu Bai Ya-Nan Lv Mei Zeng Pei-Yao Jia Hu-Na Lu Yi-Bo Zhu Sheng Li Ying-Ying Cui Yun-Xia Luan Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) Genes <i>Thermobia domestica</i> reference genes expression stability quantitative real-time PCR RNA <i>interference</i> |
| title | Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) |
| title_full | Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) |
| title_fullStr | Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) |
| title_full_unstemmed | Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) |
| title_short | Selection of Reference Genes for Normalization of Gene Expression in <i>Thermobia domestica</i> (Insecta: Zygentoma: Lepismatidae) |
| title_sort | selection of reference genes for normalization of gene expression in i thermobia domestica i insecta zygentoma lepismatidae |
| topic | <i>Thermobia domestica</i> reference genes expression stability quantitative real-time PCR RNA <i>interference</i> |
| url | https://www.mdpi.com/2073-4425/12/1/21 |
| work_keys_str_mv | AT yubai selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT yananlv selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT meizeng selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT peiyaojia selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT hunalu selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT yibozhu selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT shengli selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT yingyingcui selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae AT yunxialuan selectionofreferencegenesfornormalizationofgeneexpressioninithermobiadomesticaiinsectazygentomalepismatidae |