Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae)
Stable reference genes (RGs) determine the reliability of quantitative polymerase chain reaction (qPCR) analyses and it is recommended that different reference genes are used for different types of DNA and tissues. The present study aimed to screen for stable RGs for the qPCR analysis of the immune...
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Institute of Entomology, Biology Centre, Czech Academy of Science
2019-11-01
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doaj-ce64a9ff68e64477913bccfc3220f96e2021-04-16T20:36:07ZengInstitute of Entomology, Biology Centre, Czech Academy of ScienceEuropean Journal of Entomology1210-57591802-88292019-11-01116140240210.14411/eje.2019.041eje-201901-0041Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae)Xin-Chao LIU0Zheng-Xi LIDepartment of Entomology and Key Laboratory of Pest Monitoring and Green Management, MOA, College of Plant Protection, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, China; e-mails: zxli@cau.edu.cn, 1696679923@qq.comStable reference genes (RGs) determine the reliability of quantitative polymerase chain reaction (qPCR) analyses and it is recommended that different reference genes are used for different types of DNA and tissues. The present study aimed to screen for stable RGs for the qPCR analysis of the immune responses of the whitefly Bemisia tabaci to the Wolbachia wMel strain from Drosophila melanogaster. A total of eight candidate RGs were evaluated using five different methods, i.e., Coefficient of Variation analysis, GeNorm, NormFinder, BestKeeper and ΔCt. The stability of these RGs was assessed for both genomic DNA (gDNA) and complementary DNA (cDNA). The results indicate that β-actin (Actin) and elongation factor 1 alpha (EF-1α) were the most stable RGs for gDNA, whereas 18S rRNA (18S) and glyceraldehyde phosphate dehydrogenase (GAPDH) were the least stable; in contrast, Actin and GAPDH were the most stable for cDNA, whereas RPL29 and ATPase were the least stable. The effectiveness of the most stable RGs was then validated against the least stable using qPCR analysis of the titre of wMel (gDNA) and the transcriptional responses of the antimicrobial peptide Alo-3-like and the phosphatidylinositol-bisphosphate 3-kinase catalytic subunit delta isoform (cDNA) to wMel transfection. The results support the notion that reliable RGs are essential for a qPCR analysis of samples of both gDNA and cDNA.https://www.eje.cz/artkey/eje-201901-0041_screening_for_stable_internal_reference_genes_for_quantitative_pcr_analysis_of_wolbachia-host_interactions_in_w.phphomopteraaleyrodidaebemisia tabacireference genequantitative pcrwolbachia transfection |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xin-Chao LIU Zheng-Xi LI |
spellingShingle |
Xin-Chao LIU Zheng-Xi LI Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) European Journal of Entomology homoptera aleyrodidae bemisia tabaci reference gene quantitative pcr wolbachia transfection |
author_facet |
Xin-Chao LIU Zheng-Xi LI |
author_sort |
Xin-Chao LIU |
title |
Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) |
title_short |
Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) |
title_full |
Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) |
title_fullStr |
Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) |
title_full_unstemmed |
Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae) |
title_sort |
screening for stable internal reference genes for quantitative pcr analysis of wolbachia-host interactions in whitefly bemisia tabaci (homoptera: aleyrodidae) |
publisher |
Institute of Entomology, Biology Centre, Czech Academy of Science |
series |
European Journal of Entomology |
issn |
1210-5759 1802-8829 |
publishDate |
2019-11-01 |
description |
Stable reference genes (RGs) determine the reliability of quantitative polymerase chain reaction (qPCR) analyses and it is recommended that different reference genes are used for different types of DNA and tissues. The present study aimed to screen for stable RGs for the qPCR analysis of the immune responses of the whitefly Bemisia tabaci to the Wolbachia wMel strain from Drosophila melanogaster. A total of eight candidate RGs were evaluated using five different methods, i.e., Coefficient of Variation analysis, GeNorm, NormFinder, BestKeeper and ΔCt. The stability of these RGs was assessed for both genomic DNA (gDNA) and complementary DNA (cDNA). The results indicate that β-actin (Actin) and elongation factor 1 alpha (EF-1α) were the most stable RGs for gDNA, whereas 18S rRNA (18S) and glyceraldehyde phosphate dehydrogenase (GAPDH) were the least stable; in contrast, Actin and GAPDH were the most stable for cDNA, whereas RPL29 and ATPase were the least stable. The effectiveness of the most stable RGs was then validated against the least stable using qPCR analysis of the titre of wMel (gDNA) and the transcriptional responses of the antimicrobial peptide Alo-3-like and the phosphatidylinositol-bisphosphate 3-kinase catalytic subunit delta isoform (cDNA) to wMel transfection. The results support the notion that reliable RGs are essential for a qPCR analysis of samples of both gDNA and cDNA. |
topic |
homoptera aleyrodidae bemisia tabaci reference gene quantitative pcr wolbachia transfection |
url |
https://www.eje.cz/artkey/eje-201901-0041_screening_for_stable_internal_reference_genes_for_quantitative_pcr_analysis_of_wolbachia-host_interactions_in_w.php |
work_keys_str_mv |
AT xinchaoliu screeningforstableinternalreferencegenesforquantitativepcranalysisofwolbachiahostinteractionsinwhiteflybemisiatabacihomopteraaleyrodidae AT zhengxili screeningforstableinternalreferencegenesforquantitativepcranalysisofwolbachiahostinteractionsinwhiteflybemisiatabacihomopteraaleyrodidae |
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