Reference gene selection and evaluation for expression analysis using qRT-PCR in <i>Galeruca daurica</i> (Joannis)

Tan, Yibo; Zhou, Xiaomao; Pang, Bao-Ping

doi:10.1017/s0007485316000948

Cited by 38 publications

(42 citation statements)

References 31 publications

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“…The mRNA expression levels of GdHsp10 and GdHsp60 were examined using a SYBR Green ® real-time PCR assay (SYBR PrimeScrip™ RT-PCR Kit II, Takara) with FTC-3000P Real-time PCR (Funglyn Biotech, Canada). The succinate dehydrogenase (SDHA) gene was confirmed stably expressed among different treatments in G. daurica (Tan et al, 2017), and was used as an internal control. Primers for qPCR were designed using online software Primer 3.0 (http://primer3.ut.ee/) and listed in table 1.…”

Section: Quantitative Analysis Of Gdhsp10 and Gdhsp60 Mrna Expressionmentioning

confidence: 99%

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis

Tan

Zhang

Huo

et al. 2017

Bull. Entomol. Res.

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Galeruca daurica (Joannis) is a new outbreak pest in the Inner Mongolia grasslands in northern China. Heat shock protein 10 and 60 (Hsp10 and Hsp60) genes of G. daurica, designated as GdHsp10 and GdHsp60, were cloned by rapid amplification of cDNA ends techniques. Sequence analysis showed that GdHsp10 and GdHsp60 encoded polypeptides of 104 and 573 amino acids, respectively. Sequence alignment and phylogenetic analysis clearly revealed that the amino acids of GdHsp10 and GdHsp60 had high homology and were clustered with other Hsp10 and Hsp60 genes in insects which are highly relative with G. daurica based on morphologic taxonomy. The mRNA expression analysis by real-time PCR revealed that GdHsp10 and GdHsp60 were expressed at all development stages and in all tissues examined, but expressed highest in eggs and in adults' abdomen; both heat and cold stresses could induce mRNA expression of GdHsp10 and GdHsp60 in the 2nd instar larvae; the two Hsp genes were expressed from high to low with the extension of treatment time in G. daurica eggs exposed to freezing point. Overall, our study provides useful information to understand temperature stress responses of Hsp60 and Hsp10 in G. daurica, and provides a basis to further study functions of Hsp60/Hsp10 relative to thermotolerance and cold hardiness mechanism.

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Section: Quantitative Analysis Of Gdhsp10 and Gdhsp60 Mrna Expressionmentioning

confidence: 99%

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis

Tan

Zhang

Huo

et al. 2017

Bull. Entomol. Res.

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“…Quantitative real-time PCR (qRT-PCR) is a valuable tool for gene expression studies due to its high sensitivity, specificity and convenience in high throughput analyses 26 . In general, when the technique is used to analyze gene expressions, reference genes (RGs) are needed to normalize nonspecific variations or errors that can be caused by sample quantity, variations in efficiency of RNA extraction, cDNA concentration, primer performance, PCR efficiency and experimental precision 27 , 28 . The optimal number of RGs is another normalization factor because one single reference gene is not enough to normalize gene expression data 28 , 29 .…”

Section: Introductionmentioning

confidence: 99%

Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR

Tang

Duan

et al. 2017

Sci Rep

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Cryptolestes ferrugineus is a serious pest of stored grain and has developed high levels of resistance to phosphine fumigants in many countries. Measuring differences in expression levels of certain ‘resistant’ genes by quantitative real-time PCR (qRT-PCR) may provide insights into molecular mechanisms underlying resistance to phosphine in C. ferrugineus, but reliable qRT-PCR results depend on suitable reference genes (RGs). We evaluated the stability of nine candidate RGs across different developmental stages and phosphine strains of C. ferrugineus, using four softwares. The results showed that RPS13 and EF1α were the most stable RGs, whereas α-TUB was the least under developmental stages. Across the different strains, RPS13 and γ-TUB were the most stable RGs, whereas CycA and GAPDH were the least. We confirmed the reliability of the selected RGs by qRT-PCR analyses of the mitochondrial cox1 gene. Expression of cox1 was not significantly different in the phosphine-resistant strain compared with the phosphine-susceptible strain, but three mitochondrial genes (nad3, atp6 and cob) were significantly down-regulated. These results suggest that alterations in the expressions of these three genes may be associated with phosphine resistance in C. ferrugineus. The findings will facilitate future functional genomics studies on the development and phosphine resistance in C. ferrugineus.

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“…However, RT-qPCR requires normalization by reference genes to offset variation among samples processed from different tissues [36]. In recent years, with the gradual deepening of insect molecular biology research, it is becoming increasingly necessary to select appropriate reference genes to evaluate many biological processes or important functional genes in insects [2,3,22,24,29,[37][38][39][40][41][42][43][44][45][46][47]. Regardless, there is no study to date on reference genes in E. https://doi.org/10.1371/journal.pone.0228308.g002…”

Section: Discussionmentioning

confidence: 99%

“…Eucryptorrhynchus scrobiculatus Motschulsky (Coleoptera: Curculionidae), an important wood-boring pest of the tree-of-heaven Ailanthus altissima Swingl [1][2][3] that is widely distributed in 21 provinces in China [4], is one of the most damaging invasive insects in this country. E. scrobiculatus larvae feed on A. altissima roots, and adults feed on A. altissima twigs, which usually kills the trees within 3-5 years [5].…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

2020

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Eucryptorrhynchus scrobiculatus is an important wood-boring pest of Ailanthus altissima in China, where it causes a large number of these trees to weaken or even die. To identify genes related to economic traits or specific cellular processes in E. scrobiculatus, gene expression in multiple tissue/organ samples is commonly surveyed, and reference genes are required in this process as a control for normalization. In the present study, 18 candidate reference genes from E. scrobiculatus were identified, and the expression levels of these reference genes were estimated through quantitative real-time PCR. Differences in expression levels were analyzed with four algorithms (geNorm, NormFinder, BestKeeper and delta Ct) and comprehensively with RefFinder. With the most stable levels of expression in different tissues, RPL13, RPS3 and RPL36 were determined to be suitable for use as candidate reference genes. Moreover, the expression profile of one target gene (glycoside hydrolase family 45, GH45) confirmed the reliability of the selected candidate reference genes. This study provides the first set of suitable candidate reference genes for gene expression studies in E. scrobiculatus, and the findings will facilitate subsequent transcriptomics and functional gene research on this pest.

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Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

Cited by 38 publications

References 31 publications

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis

Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

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