Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae)

Title:

Screening for stable internal reference genes for quantitative PCR analysis of Wolbachia-host interactions in whitefly Bemisia tabaci (Homoptera: Aleyrodidae)

Creator:

Liu, Xin-Chao and Li, Zheng-Xi

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:fe0d61fe-7b24-4070-bcfd-cc4ea46f62f1
uuid:fe0d61fe-7b24-4070-bcfd-cc4ea46f62f1
doi:10.14411/eje.2019.041

Subject:

Homoptera, Aleyrodidae, Bemisia tabaci, reference gene, quantitative PCR, and Wolbachia transfection

Type:

model:article and TEXT

Format:

počítač and online zdroj

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.

Language:

English

Rights:

http://creativecommons.org/publicdomain/mark/1.0/
policy:public

Coverage:

402-412

Source:

European Journal of Entomology | 2019 Volume:116 | Number:1

Harvested from:

CDK

Metadata only:

false