Abstract
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.
Highlights
Real-time quantitative PCR is a commonly used technique for gene quantitation at both genomic DNA and complementary DNA levels, which recently has been used in many studies (Artico et al, 2010; Derveaux et al, 2010; Hindson et al, 2013)
Based on quantitative PCR (qPCR) analysis of genomic DNA (gDNA) and complementary DNA (cDNA) samples taken from transinfected B. tabaci adults and the different algorithms (GeNorm, NormFinder, BestKeeper or ΔCt method), stabilities were assigned to each reference genes (RGs) and the geomeans of these stabilities calculated, which were used to determine their combined ranking (Table 2)
The results indicated that ATPase and EF-1α were the two most stable RGs for gDNA templates and ribosomal protein L29 (RPL29) and glyceraldehyde phosphate dehydrogenase (GAPDH) the two least stable RGs; in contrast, 18S and Actin were the two most stable RGs for cDNA templates, whereas EF-1α and ATPase were the two least stable RGs
Summary
Real-time quantitative PCR (qPCR) is a commonly used technique for gene quantitation at both genomic DNA (gDNA) and complementary DNA (cDNA) levels, which recently has been used in many studies (Artico et al, 2010; Derveaux et al, 2010; Hindson et al, 2013). Several algorithms have been developed for evaluating the stability of RGs, including the Coefficient of Variation (Boda et al, 2009), GeNorm (Vandesompele et al, 2002), NormFinder (Andersen et al, 2004), BestKeeper (Pfaffl et al, 2004) and the ΔCt method (Silver et al, 2006). These algorithms have proved to be helpful in the screening for stable RGs in a variety of organisms (Freitag et al, 2018; Meng et al, 2019; Xie et al, 2019)
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