Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

Chang, Ya-Wen; Chen, Jingyun; Lu, Ming‐Xing; Gao, Yuan; Tian, Zi-Hua; Gong, Wei-Rong; Zhu, Wei; Du, Yu‐Zhou

doi:10.1371/journal.pone.0181862

Cited by 39 publications

(45 citation statements)

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“…In this study, 18S was ranked as the most stable reference gene across developmental stages and various adult tissues of B. odoriphaga. Similar results concerning developmental stage were obtained in Liriomyza trifolii, Helicoverpa armigera and Sesamia inferens (Chandra et al 2014;Sun et al 2015;Chang et al 2017). A previous study showed that 18S was the most reliable gene for normalization in RT-qPCR when investigating gene expression in different organs of Rhodnius prolixus (Majerowicz et al 2011).…”

Section: Discussionsupporting

confidence: 76%

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Selection of reference genes for quantitative real‐time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae)

Tang

Dai

Zhang

2019

Entomological Science

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Selecting stable reference genes for reverse transcription quantitative real‐time polymerase chain reaction analysis is critically important for gene expression. Bradysia odoriphaga is the most serious pest of Chinese chives, but our knowledge of its genetics is limited. In the present study, five housekeeping genes (β‐Actin, α‐Tubulin, RPS7, GAPDH and 18S rRNA) were cloned from B. odoriphaga using the combined techniques of reverse transcription polymerase chain reaction with rapid amplification of cDNA ends. The five genes, together with RPS15, were quantified for transcription stability in B. odoriphaga under various experimental conditions. Their expression stability was evaluated using four different algorithms (a comparative ΔCt method, GeNorm, NormFinder and BestKeeper). Additionally, we used an online Web‐based tool (RefFinder) to assign an overall final rank to each candidate gene. These analyses identified 18S, RPS15 and RPS7 as the most stable reference genes across developmental stages. 18S, RPS7 and Tubulin were the most stable reference genes for monitoring gene expression across different tissues of adults. RPS7, GAPDH and Tubulin were selected as the best reference genes across different larval tissues. GAPDH and RPS15 were selected to normalize gene expression for experiments of insecticide treatments. Actin and GAPDH are considered suitable reference genes in experiments of temperature treatments. Actin and Tubulin are considered suitable reference genes for starvation experiments. This study provides an important supplement of suitable reference genes for B. odoriphaga and these results provide clues toward the understanding of the genes’ biological functions in B. odoriphaga.

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Section: Discussionsupporting

confidence: 76%

“…; Chang et al . ). A previous study showed that 1 8S was the most reliable gene for normalization in RT‐qPCR when investigating gene expression in different organs of Rhodnius prolixus (Majerowicz et al .…”

Section: Discussionmentioning

confidence: 97%

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae)

Tang

Dai

Zhang

2019

Entomological Science

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“…lugens , Liriomyza trifolii , B . tabaci , Tetranychus cinnabarinus , Monochamus alternatus and Sogatella furcifera [ 48 – 51 ]. In addition, α-TUB was ranked as the most stable RG in different tissues of C .…”

Section: Discussionmentioning

confidence: 99%

Reference genes selection for quantitative gene expression studies in tea green leafhoppers, Empoasca onukii Matsuda

Zhang

Huang

et al. 2018

PLoS ONE

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Empoasca onukii Matsuda is one of the most devastating pests of the tea plant (Camellia sinensis). Still, the presumed expression stability of its reference genes (RGs) has not been analyzed. RGs are essential for accurate and reliable gene expression analysis, so this absence has hampered the study of the insect’s molecular biology. To find candidate RGs for normalizing gene expression data, we cloned ten common housekeeping genes from E. onukii. Using the ΔCt method, geNorm, NormFinder and BestKeeper, we screened the RGs that were appropriate for quantifying the mRNA transcription of cellular responses under five experimental conditions. We identified the combinations of α-TUB and G6PDH, α-TUB and UBC, two RGs (α-TUB and β-TUB1) or three RGs (α-TUB, RPL13 and GAPDH), AK and UBC, or RPL13 and α-TUB as the best for analyzing gene expression in E. onukii adults of both sexes in different tissues, nymphs at different developmental stages, nymphs exposed to different temperatures or nymphs exposed to photoperiod stress. Finally, the E. onukii cysteine proteinase (Eocyp) was chosen as the target gene to validate the rationality of the proposed RGs. In conclusion, our study suggests a series of RGs with which to study the gene expression profiles of E. onukii that have been manipulated (biotically or abiotically) using reverse transcription quantitative polymerase chain reaction. The results offer a solid foundation for further studies of the molecular biology of E. onukii.

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“…Primer Express v 2.0 software (Thermo Fisher Scientific) was used for primer design. PCR primer sequences were designed according to the reference literature 18 as follows: PBX3 forward, 5′-CTCCCAAATTCTGGGGACATG-3′ and reverse, 5′-ATCCACCTGTGACTGCACATTG-3′; 11 and GAPDH forward, 5′-AGGCTGTTGGCAAAGTGATTC-3′ and reverse, 5′-CTTTTCCCAAACGCACAGTCA-3′.…”

Section: Methodsmentioning

confidence: 99%

PBX3 is associated with proliferation and poor prognosis in patients with cervical cancer

Sun

Liu

et al. 2017

OTT

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Pre-B-cell leukemia homeobox 3 (PBX3) is upregulated in various malignancies; however, the role of PBX3 in cervical cancer (CC) is unknown. The purpose of this study was to explore the expression characteristics, clinicopathological significance, and molecular biological function of PBX3 in CC. The expression levels of PBX3 were analyzed in CC cell lines and tumor specimens by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining. The clinicopathological characteristics associated with PBX3 expression were evaluated. An RNA interference approach was employed to suppress PBX3 expression in CC in vitro and in vivo, determine its role in cell proliferation and analyze its molecular function. We found that PBX3 expression was significantly upregulated in CC cell lines and clinical specimens compared with normal cells and adjacent nontumorous cervical tissues. PBX3 was an independent predictive factor of poor prognosis, and its expression was correlated with tumor diameter, pathological grading, lymph node metastasis, invasion depth, vascular invasion, and clinical stage of CC. Multivariate analysis suggested that PBX3 expression may represent an independent prognostic indicator of the survival of CC patients. CC patients with high PBX3 expression exhibited reduced overall survival compared with those with low PBX3 expression. Additionally, stable downregulation of PBX3 expression in CC cell lines suppressed cell proliferation and decreased p-AKT protein expression levels in vitro. Similarly, in vivo assays demonstrated that PBX3 downregulation in CC cells markedly inhibited tumor size and weight. Overall, we demonstrated that PBX3 can promote CC cell proliferation via the AKT signaling pathway and that it may serve as a prognostic marker. Our data indicate that inactivation of PBX3 may be an effective clinical treatment for CC.

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Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

Cited by 39 publications

References 50 publications

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae)

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae)

Reference genes selection for quantitative gene expression studies in tea green leafhoppers, Empoasca onukii Matsuda

PBX3 is associated with proliferation and poor prognosis in patients with cervical cancer

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