Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae)

Abstract

Quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR) is preferred for gene expression analysis in living organisms. Currently, it is a valuable tool for biological and ecological studies as it provides a relatively straightforward way to assess the relevance of transcriptional regulation under developmental and stress tolerance conditions. However, studies have shown that some commonly used reference genes varied among different experimental treatments, thus, systematic evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of arthropods. The aim of this study is to identify the suitable reference genes for RT-qPCR experiments involving various developmental stages and/or under abiotic stresses in citrus red mite Panonychus citri, a key pest in citrus orchards worldwide. GeNorm, NormFinder, and Bestkeeper software analysis indicates that elongation factor-1 alpha (ELF1A), RNA polymerase II largest subunit, alpha tublin, and glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are the most stable reference genes in various developmental stages, meanwhile, ELF1A and GAPDH were the most stable reference genes under various abiotic stresses. Furthermore, this study will serve as a resource to screen reference genes for gene expression studies in any other spider mite species.

Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in <I>Amblyomma maculatum</I> (Acari: Ixodidae)

“…Additionally, when all samples were examined together BestKeeper also ranked GAPDH as the most stable housekeeping gene; however, while the SD of GAPDH, 1.21, was the lowest of all Þve candidate reference genes, it was outside of the suggested threshold. It is difÞcult to identify which is actually the most stably expressed reference gene when examining all developmental stages and tissues based on the BestKeeper analysis, as it has been previously reported that reference gene stability should be based on both the r value and SD value (Takle et al 2007, MarouÞ et al 2010, Niu et al 2012.…”

“…Traditional housekeeping genes such as ␤-actin and GAPDH have not always been validated in all experimental conditions in arthropods and later gene expression studies have shown that other genes including ␣-tubulin (Bactrocera dorsalis); elongation factor 1-á (EF1␣), RNA polymerase II large subunit, ␣-tubulin, GAPDH (Panonychus citri); and EF1␣ (Rhipicephalus appendiculatus and R. microplus) may be more reliable for use as a reference genes in qRT-PCR analysis (Nijhof et al 2009, Shen et al 2010, Niu et al 2012. Interestingly, Ixodes scapularis (Say) ␤-actin is routinely used in qRT-PCR experiments (Pedra et al 2006, Sukumaran et al 2006, Karim et al 2010, however, an elegant study showed that Anaplasma phagocytophilum induces Actin phosphorylation in I. scapularis .…”

Differential Analysis of the Cytochrome p450 Acaricide-Resistance Genes inPanonychus citri(Trombidiformes: Tetranychidae) Strains

“…Multiple specific Primers (Table 2) were designed by using Primer Premier 5.0 software (PREMIER Biosoft International, California). The expression study was performed on an Mx3000P TM Multiple Quantitative PCR System (Stratagene, La Jolla, California, USA) with ELF1A, elongation factor-1 alpha, used as reference gene (Niu et al 2012). The reaction system and reaction parameters were same as Niu et al (2012) described earlier.…”

“…The expression study was performed on an Mx3000P TM Multiple Quantitative PCR System (Stratagene, La Jolla, California, USA) with ELF1A, elongation factor-1 alpha, used as reference gene (Niu et al 2012). The reaction system and reaction parameters were same as Niu et al (2012) described earlier. After collecting the Ct values, the relative quantitative expression of these 5 genes were calculated by the 2 -СT method (Livak & Schmittgen 2001).…”

Genes de referência de <i>Diaphorina citri</i> para estudos de expressão gênica quantitativa e seu controle por RNAi em laranja doce

“…Está envolvida em diversos processos celulares tais como mobilidade celular, contração muscular, citocinese e divisão celular (Niu et al 2012;Lu et al 2013 Helicoverpa armigera (Zhang et al 2014;Shakeel et al, 2015), Nilaparvata lugens (Yuan et al, 2014), Bemisia tabaci , Spodoptora litua (Lu et al, 2013), Frankliniella occidentalis (Zheng et al, 2014) e Sesamia inferens (Sun et al, 2015). , Helicoverpa armigera (Zhang et al, 2014;Shakeel et al, 2015), Sesamia inferens (Sun et al, 2015), Toxoptera citricida (Shang et al, 2015).…”