Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Megalurothrips usitatus (thysanoptera: thripidae)

Introduction: Gene expression analysis by reverse transcription quantitative polymerase chain reaction (qRT-PCR) has been widely used in research including insects. The selection of appropriate reference genes is the key to obtaining accurate and reliable results from qRT-PCR. However, studies on the expression stability of reference genes in Megalurothrips usitatus remain lacking.Methods: In this study, qRT-PCR was used to analyze the expression stability of candidate reference genes in M. usitatus. The expression levels of six candidate reference gene transcription of M. usitatus were analyzed. GeNorm, NormFinder, BestKeeper, and ΔCt were used to analyze the expression stability of M. usitatus treated with biological factors (developmental period treatment) and abiotic factors (light, temperature, insecticide treatment, respectively). Comprehensive stability ranking of candidate reference genes was recommended by RefFinder.Results and Discussion: Results showed that ribosomal protein S (RPS) was the most suitable expression in insecticide treatment. Ribosomal protein L (RPL) was the most suitable expression at developmental stage and light treatment, whereas elongation factor was the most suitable expression in temperature treatment. RefFinder was used to comprehensively analyze the above four treatments, and the results showed that RPL and actin (ACT) showed high stability in each treatment. Therefore, this study identified these two genes as reference genes in the qRT-PCR analysis of different treatment conditions of M. usitatus. Ourfindings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in M. usitatus

Image1_Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Megalurothrips usitatus (thysanoptera: thripidae).JPEG

Introduction: Gene expression analysis by reverse transcription quantitative polymerase chain reaction (qRT-PCR) has been widely used in research including insects. The selection of appropriate reference genes is the key to obtaining accurate and reliable results from qRT-PCR. However, studies on the expression stability of reference genes in Megalurothrips usitatus remain lacking.Methods: In this study, qRT-PCR was used to analyze the expression stability of candidate reference genes in M. usitatus. The expression levels of six candidate reference gene transcription of M. usitatus were analyzed. GeNorm, NormFinder, BestKeeper, and ΔCt were used to analyze the expression stability of M. usitatus treated with biological factors (developmental period treatment) and abiotic factors (light, temperature, insecticide treatment, respectively). Comprehensive stability ranking of candidate reference genes was recommended by RefFinder.Results and Discussion: Results showed that ribosomal protein S (RPS) was the most suitable expression in insecticide treatment. Ribosomal protein L (RPL) was the most suitable expression at developmental stage and light treatment, whereas elongation factor was the most suitable expression in temperature treatment. RefFinder was used to comprehensively analyze the above four treatments, and the results showed that RPL and actin (ACT) showed high stability in each treatment. Therefore, this study identified these two genes as reference genes in the qRT-PCR analysis of different treatment conditions of M. usitatus. Ourfindings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in M. usitatus.</p

UVA induces CatK expression in a dose-dependent manner.

<p>Cultured fibroblasts were mock irradiated or irradiated with 10/cm² UVA, 20 J/cm² UVA and 30 J/cm² UVA. Cells were harvested 48 h after UVA irradiation for total RNA and protein extraction. (A) Quantitative analysis of CatK mRNA level with real-time RT-PCR. (B) Analysis of CatK protein level by western blot using anti-CatK antibody. Blots were reprobed with anti-GAPDH to confirm equal loading. Bar graphs represent the relative band intensities (mean±SD from three independent experiments). *P<0.05 vs. untreated control, §P<0.05 vs. both other dose-treated groups.</p

effect of Jun and Fos siRNA on UVA-induced CatK expression.

<p>Cells were cotransfected at 50–70% confluence with 50 nM Jun siRNA and 50 nM Fos siRNA, or 50 nM non-targeting control siRNA (NC) according to the manufacturer's protocol. At 24 h following transfection, the medium was replaced with PBS, cells in control (C), non-targeting-siRNA transfected control (NC) and Jun and Fos siRNA transfected group (siRNA) were not irradiated, whereas cells in UVA-C, UVA-NC and UVA-siRNA were irradiated by 10 J/cm² UVA, and then incubated with fresh culture medium for an additional 48 h. (A) mRNA level of Jun and Fos in their siRNA-transfected fibroblasts. (B) protein level of Jun and Fos in their siRNA-transfected fibroblasts.(C) Effect of Jun and Fos siRNA on UVA-induced CatK mRNA expression. Total cellular RNA was isolated at 48 h after irradiation, and was analyzed by real-time RT-PCR for CatK. (D) Effect of Jun and Fos siRNA on UVA-induced CatK protein expression. Total cellular protein was harvested at 48 h after irradiation,then was analyzed by Western blot with anti-CatK antibody. *P<0.05 vs. untreated control, §P<0.05 vs. UVA-treated control.</p

Inhibitory effects of SP600125 and SB203580 on UVA-activated MAPK pathway.

<p>Cells were pretreated with 800 µM SB203580 for 1 h, mock-irradiated or irradiated with 10 J/cm² UVA, and then incubated in culture medium supplemented with MAPK inhibitors for 1.5 h. Fifteen micrograms of total cellular lysate was electrophoresed on 10% SDS polyacrylamide gels, transferred to a PVDF membrane, and immunodetected using optimal primary and secondary antibody concentrations. Western blot data are representative of three independent experiments. (A) Effects of SP600125 and SB203580 on JNK activity through detection of changes in Jun phosphorylation; (B) Effects of SP600125 and SB203580 on p38 activity through detection of changes in MAPKAPK-2 phosphorylation. *P<0.05 vs. untreated control, §P<0.05 vs.UVA-treated control.</p

Time courses for UVA-induced MAPK activation and AP-1 activity.

<p>Cells were mock-irradiated or irradiated with 10 J/cm² UVA and harvested at the indicated time points. Western blot analysis was performed and the representative data of three independent experiments were shown. Figure showed the expression of (A) JNK; (B) p38; (C) Jun; (D) Fos.</p