Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

Abstract

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

Validation of reference genes in leaf-cutting ant Atta sexdens rubropilosa in different developmental stages and tissues

“…The diversity of ant species indicates that they rank among the most successful insects. It is estimated that 40.000 ant species exist in the world, of which about 16.000 species and subspecies have already been formally described [2]. All ants are considered eusocial.…”

“…Incorrect results might be obtained due to misinterpretation of RNA transcription levels especially for low abundance gene transcripts [10,11]. To date, several works were developed to determine the reference gene in Insecta, which demonstrate that is impossible to find a universal reference gene able to covering all organism and conditions [11,12,13,14,15,16,17]. The choice of a gene as reference gene is not trivial and starts with the selection of candidate reference genes to be analyzed [18].…”

“…A good strategy for selecting potential candidate reference genes is based on previous data from species relative to the studied specie due to the high degree of similarity between genomes and the expectation of a similar expression level [9]. However, differences in stability have been verified in the analysis of a reference gene in Insecta for organisms from the same order [11,12,14,15], family [20,21] and even for those of the same genus [14,16,21]. This justifies studies to validate reference genes for an organism and experimental conditions before the analysis for precise mRNA quantification [22].…”

Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua

“…qPCR is generally characterise as an effective, sensitive, and economical methods, it has already widely applied to analyse gene expression in biological research (Overbergh 2003;Lu et al 2013;Liang et al 2014). However, there remain a number of problems have not yet been settled.…”

Bases genéticas e moleculares da resistência de <i>Spodoptera frugiperda</i> (J.E. Smith) (Lepidoptera: Noctuidae) a spinosad

“…Essas informações podem ser exploradas a partir da avaliação do padrão de herança e custo adaptativo associado à resistência, em estudos de resistência cruzada, frequência inicial de alelos da resistência em populações de campo e modelos de simulação de evolução da resistência DALY 1990, TABASHNIK, 1991 (VÉLEZ et al, 2013;JAKKA et al, 2014, LU et al, 2014 Nos últimos anos observou-se uma grande expansão da utilização de análises moleculares para caracterização de mecanismos de resistência de insetos a inseticidas e toxinas Bt, caracterizando genes e enzimas a fim de se elucidar os aspectos fisiológicos e moleculares envolvidos nessa resistência (RANSON et al, 2002). Os principais mecanismos associados à resistência de insetos a inseticidas são o aumento da taxa de detoxificação metabólica, a redução da sensibilidade do sítio de ação e a redução da penetração cuticular do produto (GEORGHIOU, 1972;SCOTT, 1990;SCOTT, 1995 O segundo mecanismo mais comum tem sido a mudança da sensibilidade do sítio de ação do inseticida.…”