Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm &amp;lt;italic&amp;gt;Bombyx mori&amp;lt;/italic&amp;gt;

Ran, Pixin; Zhai, Yuanfen; Ding, Hua; Di, Tianyuan; Zhang, Ting; Li, Bing; Shen, Weide; Wei, Zheng-Guo

doi:10.1093/abbs/gms040

Cited by 27 publications

(20 citation statements)

References 21 publications

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“…Expression levels of all genes were normalized to the levels of sigA gene transcripts 55 . The degrees of change in expression level were calculated using the 2 −ΔΔ Ct method 56 .…”

Section: Methodsmentioning

confidence: 99%

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Zhang

Jiang

et al. 2017

Sci Rep

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Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.

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“…Expression levels of all genes were normalized to the levels of sigA gene transcripts 55 . The degrees of change in expression level were calculated using the 2 −ΔΔ Ct method 56 .…”

Section: Methodsmentioning

confidence: 99%

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Zhang

Jiang

et al. 2017

Sci Rep

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“…Ran Peng [37] et al detected seven commonly used reference genes (ACT,GAPDH, 28SrRNA, RPL3, a-Tubulin, UBC and TBP) in different development stages of Bombyx mori. ACT3, GAPDH and a-Tubulin in the mid-gut, α-tubulin, UBC and TBP in the fat body as well as a-Tubulin, ACT3 and UBC in the Malpighian tubule were identified as the most stable genes.…”

Section: Research Of Reference Genes For Insectsmentioning

confidence: 99%

Research Progress on Reference Genes of Insect for Quantitative Real-time Reverse Transcription PCR (RT-qPCR)

Shi¹,

Hu²,

Zhang³

2015

ujar

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Quantitative real-time reverse transcription PCR (RT-qPCR) has become the most important method for the quantification of mRNA transcription levels owing to its, specificity, sensitivity, reproducibility, and efficiency. In order to avoid sample-to-sample and run-to-run variations particularly in RNA extraction, RNA quality and cDNA reverse transcription level ,etc. it is necessary to use housekeeping gene which stably expressing as reference gene. Ideally, the housekeeping gene should not be regulated or influenced by the experimental procedure or co-regulated with the target gene. Studies insect models have shown that the expression levels of commonly used reference genes can differ among different tissue, organ types or physiological conditions. However, improper selection of reference genes will result in inaccurate calculation results and consequently obscure actual biological differences among samples, even opposite conclusion. Therefore, reference genes which specific stably expression in each experimental system should be selected in different insects and different experiments. This review aims to provide research achievements of domestic and foreign scholars on insect reference genes, which provide great promise for the future.

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“…Samples were run in triplicate and contained 1.5 µL 2 µM specific primers, 7.5 µL SYBR Premix Ex Tag II (Tli RNaseH Plus, Takara; 2×) and 2 µL cDNA; RNase-free water was added to achieve a final volume of 15 µL. The housekeeping gene Ribosomal protein L3 ( RPL3 ) was used as an RNA loading control, as previously described [17] . Data were transformed and analyzed according to the 2 −ΔΔCt method [18] and ratios after normalization were expressed as fold-changes compared to control samples.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Analysis of Neonatal Larvae after Hyperthermia-Induced Seizures in the Contractile Silkworm, Bombyx mori

et al. 2014

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The ability to respond quickly and efficiently to transient extreme environmental conditions is an important property of all biota. However, the physiological basis of thermotolerance in different species is still unclear. Here, we found that the cot mutant showed a seizure phenotype including contraction of the body, rolling, vomiting gut juice and a momentary cessation of movement, and the heartbeat rhythm of the dorsal vessel significantly increases after hyperthermia. To comprehensively understand this process at the molecular level, the transcriptomic profile of cot mutant, which is a behavior mutant that exhibits a seizure phenotype, was investigated after hyperthermia (42°C) that was induced for 5 min. By digital gene expression profiling, we determined the gene expression profile of three strains (cot/cot ok/ok, +/+ ok/ok and +/+ +/+) under hyperthermia (42°C) and normal (25°C) conditions. A Venn diagram showed that the most common differentially expressed genes (DEGs, FDR<0.01 and log2 Ratio≥1) were up-regulated and annotated with the heat shock proteins (HSPs) in 3 strains after treatment with hyperthermia, suggesting that HSPs rapidly increased in response to high temperature; 110 unique DEGs, could be identified in the cot mutant after inducing hyperthermia when compared to the control strains. Of these 110 unique DEGs, 98.18% (108 genes) were up-regulated and 1.82% (two genes) were down-regulated in the cot mutant. KEGG pathways analysis of these unique DEGs suggested that the top three KEGG pathways were “Biotin metabolism,” “Fatty acid biosynthesis” and “Purine metabolism,” implying that diverse metabolic processes are active in cot mutant induced-hyperthermia. Unique DEGs of interest were mainly involved in the ubiquitin system, nicotinic acetylcholine receptor genes, cardiac excitation–contraction coupling or the Notch signaling pathway. Insights into hyperthermia-induced alterations in gene expression and related pathways could yield hints for understanding the relationship between behaviors and environmental stimuli (hyperthermia) in insects.

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Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm <italic>Bombyx mori</italic>

Cited by 27 publications

References 21 publications

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Research Progress on Reference Genes of Insect for Quantitative Real-time Reverse Transcription PCR (RT-qPCR)

Transcriptome Analysis of Neonatal Larvae after Hyperthermia-Induced Seizures in the Contractile Silkworm, Bombyx mori

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