Identification and Validation of Reference Genes for RT-qPCR Normalization in Nauphoeta cinerea (Olivier, 1789) (Blattodea, Blaberidae)

Yıl 2022, Cilt: 6 Sayı: 1, 62 - 72, 31.03.2022

Kübra Özcan Ayşe Nur Pektaş Şeyda Berk

https://doi.org/10.30516/bilgesci.1067570

Cited By: 2

Öz

Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows large-scale analysis of very small changes in gene expression. For the calculation of gene expression, such as the delta-delta Ct method, different PCR primer efficiencies (E) may affect the result, as PCR primer yields are assumed to be comparable for the gene of interest and housekeeping gene. Therefore, identification of a suitable reference gene for data normalization is an important step in the development of qPCR assays. Furthermore, accurate and reliable results depend on the use of stable reference genes for normalization. The aim of the current study is the identification and validation of a set of six housekeeping genes (GADPH, RPS18, α-TUB, EF1α, ArgK, and ACTB) in cockroach species Nauphoeta cinerea adults using five different algorithms (ΔCt method, Bestkeeper, geNorm, Normfinder and RefFinder) to evaluate the stability of selected reference genes expression. Our results show that α-Tub use provides accurate normalization of gene expression levels in N. cinerea adults. In addition, since the GADPH is selected as the second most stable reference gene, GADPH can be also used for transcript analysis N. cinerea adults. Our study also showed that ACTB (β-actin) should not be used for normalizing transcript levels when examining N. cinerea adults. Additionally, validation studies for reference genes in cockroaches are very few (only one) in the literature. Therefore, the results highlight the need for validation of reference genes under biotic and abiotic conditions in q-RT-PCR studies in cockroaches.

Anahtar Kelimeler

RT-qPCR, reference genes, validation, normalization, Nauphoeta cinerea

Kaynakça

• Adedara, I. A., Rosemberg, D. B., Souza, D. O., Farombi, E. O., Aschner, M., and Rocha, J. B. (2016). Neuroprotection of luteolin against methylmercury-induced toxicity in lobster cockroach Nauphoeta cinerea. Environ Toxicol Pharmacol 42, 243-251.
• Adedara, I. A., Rosemberg, D. B., Souza, D. O., Kamdem, J. P., Farombi, E. O., Aschner, M., and Rocha, J. B. (2015). Biochemical and behavioral deficits in the lobster cockroach Nauphoeta cinerea model of methylmercury exposure. Toxicol Res 4, 442-451.
• Altincicek, B., Knorr, E., and Vilcinskas, A. (2008). Beetle immunity: Identification of immune-inducible genes from the model insect Tribolium castaneum. Developmental & Comparative Immunology 32, 585-595.
• An, X.-k., Hou, M.-l., and Liu, Y.-d. (2016). Reference gene selection and evaluation for gene expression studies using qRT-PCR in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae). J Econ Entomol 109, 879-886.
• Andersen, C. L., Jensen, J. L., and Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer research 64, 5245-5250. Bell, W. J., Roth, L. M., and Nalepa, C. A. (2007). Cockroaches: ecology, behavior, and natural history: JHU Press).
• Berk, S., and Pektas, A. (2020). Selection and Validation of Reference Genes for Quantitative Real-time PCR in the Mealworm Beetle, Tenebrio molitorL. (Coleoptera: Tenebrionidae). IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) 13, 44-50.
• Bohn, H., Picker, M., Klass, K.-D., and Colville, J. (2010). A jumping cockroach from South Africa, Saltoblattella montistabularis, gen. nov., spec. nov.(Blattodea: Blattellidae). Arthropod Systematics and Phylogeny 68, 53-69.
• Bouchebti, S., Durier, V., Pasquaretta, C., Rivault, C., and Lihoreau, M. (2016). Subsocial Cockroaches Nauphoeta cinerea Mate Indiscriminately with Kin Despite High Costs of Inbreeding. PLoS One 11, e0162548.
• Bustin, S. A., Benes, V., Nolan, T., and Pfaffl, M. W. (2005). Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol 34, 597-601.
• Chandna, R., Augustine, R., and Bisht, N. C. (2012). Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One 7, e36918.
• Chang, Y.-W., Chen, J.-Y., Lu, M.-X., Gao, Y., Tian, Z.-H., Gong, W.-R., Zhu, W., and Du, Y.-Z. (2017). Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae). PloS one 12, e0181862.
• Chapuis, M.-P., Tohidi-Esfahani, D., Dodgson, T., Blondin, L., Ponton, F., Cullen, D., Simpson, S. J., and Sword, G. A. (2011). Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust. BMC molecular biology 12, 1-11.
• Feuer, R., Vlaic, S., Arlt, J., Sawodny, O., Dahmen, U., Zanger, U. M., and Thomas, M. (2015). LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR) Data as an Alternative to Reference Gene Based Methods. PLoS One 10, e0135852.
• Fu, W., Xie, W., Zhang, Z., Wang, S., Wu, Q., Liu, Y., Zhou, X., Zhou, X., and Zhang, Y. (2013a). Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae). International Journal of Biological Sciences 9, 792.
• Fu, W., Xie, W., Zhang, Z., Wang, S., Wu, Q., Liu, Y., Zhou, X., Zhou, X., and Zhang, Y. (2013b). Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae). Int J Biol Sci 9, 792-802.
• Galiveti, C. R., Rozhdestvensky, T. S., Brosius, J., Lehrach, H., and Konthur, Z. (2010). Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR. RNA (New York, NY) 16, 450-461.
• García-Reina, A., Rodríguez-García, M. J., and Galián, J. (2018). Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge. Scientific reports 8, 10743.
• Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996). Real time quantitative PCR. Genome research 6, 986-994.
• Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., and Vandesompele, J. (2007). qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome biology 8, 1-14.
• Huggett, J., Dheda, K., Bustin, S., and Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and immunity 6, 279-284.
• Koramutla, M. K., Aminedi, R., and Bhattacharya, R. (2016). Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt). Scientific reports 6, 25883.
• Li, R., Xie, W., Wang, S., Wu, Q., Yang, N., Yang, X., Pan, H., Zhou, X., Bai, L., Xu, B., et al. (2013). Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS One 8, e53006.
• Li, Z., Yang, L., Wang, J., Shi, W., Pawar, R. A., Liu, Y., Xu, C., Cong, W., Hu, Q., Lu, T., et al. (2010). beta-Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum. Fish & shellfish immunology 29, 89-93.
• Liang, P., Guo, Y., Zhou, X., and Gao, X. (2014). Expression profiling in Bemisia tabaci under insecticide treatment: indicating the necessity for custom reference gene selection. PLoS One 9, e87514.
• Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif) 25, 402-408.
• Lü, J., Yang, C., Zhang, Y., and Pan, H. (2018). Selection of Reference Genes for the Normalization of RT-qPCR Data in Gene Expression Studies in Insects: A Systematic Review. Frontiers in physiology 9, 1560.
• Marchal, E., Hult, E. F., Huang, J., and Tobe, S. S. (2013). Sequencing and validation of housekeeping genes for quantitative real-time PCR during the gonadotrophic cycle of Diploptera punctata. BMC Research Notes 6, 237.
• Pabinger, S., Rödiger, S., Kriegner, A., Vierlinger, K., and Weinhäusel, A. (2014). A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomolecular Detection and Quantification 1, 23-33.
• Pan, H., Yang, X., Siegfried, B. D., and Zhou, X. (2015). A comprehensive selection of reference genes for RT-qPCR analysis in a predatory lady beetle, Hippodamia convergens (Coleoptera: Coccinellidae). PloS one 10, e0125868.
• Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic acids research 29, e45.
• Pfaffl, M. W., Tichopad, A., Prgomet, C., and Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnology letters 26, 509-515.
• Ponton, F., Chapuis, M.-P., Pernice, M., Sword, G. A., and Simpson, S. J. (2011a). Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. Journal of insect physiology 57, 840-850.
• Ponton, F., Chapuis, M. P., Pernice, M., Sword, G. A., and Simpson, S. J. (2011b). Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol 57, 840-850.
• Rodrigues, N. R., Nunes, M. E., Silva, D. G., Zemolin, A. P., Meinerz, D. F., Cruz, L. C., Pereira, A. B., Rocha, J. B., Posser, T., and Franco, J. L. (2013). Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress? Chemosphere 92, 1177-1182.
• Rodrigues, T. B., Khajuria, C., Wang, H., Matz, N., Cunha Cardoso, D., Valicente, F. H., Zhou, X., and Siegfried, B. (2014). Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera). PLoS One 9, e109825.
• Sang, W., He, L., Wang, X. P., Zhu-Salzman, K., and Lei, C. L. (2015). Evaluation of Reference Genes for RT-qPCR in Tribolium castaneum (Coleoptera: Tenebrionidae) Under UVB Stress. Environmental entomology 44, 418-425.
• Scharlaken, B., de Graaf, D. C., Goossens, K., Brunain, M., Peelman, L. J., and Jacobs, F. J. (2008). Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. J Insect Sci 8, 33.
• Schimpf, N. G., Matthews, P. G., and White, C. R. (2012). Standard metabolic rate is associated with gestation duration, but not clutch size, in speckled cockroaches Nauphoeta cinerea. Biology open 1, 1185-1191.
• Schmittgen, T. D., and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nature protocols 3, 1101-1108.
• Shakeel, M., Rodriguez, A., Tahir, U. B., and Jin, F. (2018). Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects. Biotechnology letters 40, 227-236.
• Silver, N., Best, S., Jiang, J., and Thein, S. L. J. B. m. b. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. 7, 1-9.
• Sinha, D. K., and Smith, C. M. (2014). Selection of reference genes for expression analysis in Diuraphis noxia (Hemiptera: Aphididae) fed on resistant and susceptible wheat plants. Scientific reports 4, 1-6.
• Teng, X., Zhang, Z., He, G., Yang, L., and Li, F. (2012). Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. J Insect Sci 12.
• Van Hiel, M. B., Van Wielendaele, P., Temmerman, L., Van Soest, S., Vuerinckx, K., Huybrechts, R., Broeck, J. V., and Simonet, G. (2009). Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC molecular biology 10, 1-10.
• Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology 3, Research0034. Velez, A., Wolff, M., and Gutierrez, E. (2006). Blattaria of Colombia: List and distribution of genera. Zootaxa 1210, 39-52.
• Yang, C., Pan, H., Liu, Y., and Zhou, X. (2014). Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae). PLoS One 9, e110454.
• Yang, C., Pan, H., Liu, Y., and Zhou, X. (2015a). Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae. PLoS One 10, e0120833.
• Yang, C., Pan, H., Liu, Y., and Zhou, X. (2015b). Temperature and development impacts on housekeeping gene expression in cowpea aphid, Aphis craccivora (Hemiptera: Aphidiae). PLoS One 10, e0130593.
• Yang, C., Pan, H., Noland, J. E., Zhang, D., Zhang, Z., Liu, Y., and Zhou, X. (2015c). Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific reports 5, 18201.
• Yang, C., Pan, H., Noland, J. E., Zhang, D., Zhang, Z., Liu, Y., and Zhou, X. (2015d). Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific reports 5, 1-11.
• Yang, C., Preisser, E. L., Zhang, H., Liu, Y., Dai, L., Pan, H., and Zhou, X. (2016). Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants. Frontiers in plant science 7, 1672.
• Yeung, A. T., Holloway, B. P., Adams, P. S., and Shipley, G. L. (2004). Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR. BioTechniques 36, 266-270, 272, 274-265.
• Zhang, J., Zhang, Y., Li, J., Liu, M., and Liu, Z. (2016). Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response. PLoS One 11, e0155254.
• Zhu, X., Yuan, M., Shakeel, M., Zhang, Y., Wang, S., Wang, X., Zhan, S., Kang, T., and Li, J. (2014). Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). PLoS One 9, e84730.