Abstract
Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) is considered to be the reliable and effective method for the quantitative analysis of candidate genes expression level and the verification of transcriptomic analysis, especially in species which lacking the genomic information, due to the advantages of high sensitivity and specificity, more convenience and good reproducibility (Ibanez and Tamborindeguy, 2016; Zhang et al, 2017)
Before the expression stability evaluation, eight candidate reference genes belong to four functional groups including 3 of the structure-related genes (Actin, α-Tubulin and β-Tubulin), 2 of the ribosomal protein (RPL7A and ribosomal protein L13A (RPL13A)), 2 of the protein factor (EF-1α and elongation factor 2 (EF2)), and one metabolism-related gene (GAPDH) were cloned and identified by sequencing
It was reported that the expression of reference genes is not static in different species, tissues or experimental conditions (Lu et al, 2013), so the stability verifications of reference genes are necessary before evaluating target gene expression levels by RT-qPCR
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) is considered to be the reliable and effective method for the quantitative analysis of candidate genes expression level and the verification of transcriptomic analysis, especially in species which lacking the genomic information, due to the advantages of high sensitivity and specificity, more convenience and good reproducibility (Ibanez and Tamborindeguy, 2016; Zhang et al, 2017). Reference Genes Evaluation for Insects integrity and quality, reverse transcription efficiency and primer amplification efficiency could interfere and influence the accuracy and reliability of RT-qPCR. The reference genes attracted attention and were used for the precise normalization (Zhang et al, 2015; Arya et al, 2017). The idealized reference genes which defined as the “constitutively expressed to maintain cellular function” should have the relatively stable expression under various tissues and physiological conditions (Pan et al, 2015; Chen et al, 2016). The expression levels of reference genes might vary under different experimental conditions. Selection of appropriate reference genes is the prerequisite to ensure the accuracy of experimental results (Nagy et al, 2017). Standardizing experimental results with two or more reference genes could improve the accuracy and be recommended (Shi et al, 2016)
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