The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Bustin, Stephen A.; Beneš, Vladimír; Garson, Jeremy A.; Hellemans, Jan; Huggett, Jim F.; Kubista, Mikael; Mueller, Reinhold; Nolan, Tania; Pfaffl, Michael W.; Shipley, Gregory L.; Vandesompele, Jo; Wittwer, Carl T.

doi:10.1373/clinchem.2008.112797

Cited by 13,152 publications

(10,292 citation statements)

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“…Expression of the optimal RGs should be stable under various conditions, such as in different tissues, treatment conditions, and cell lines (Robledo et al., 2014). However, due to the noncommonality of optimal RG and NF numbers in qRT‐PCR experiments, these should be determined under specific experimental conditions (Bustin et al., 2009; Vandesompele et al., 2002). Fortunately, several statistical algorithms now provide the ability to assess the expression stability of candidate RGs.…”

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Zhang

Wang

et al. 2017

Ecology and Evolution

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Changes in gene expression patterns can reflect the adaptation of organisms to divergent environments. Quantitative real‐time PCR (qRT‐PCR) is an important tool for ecological adaptation studies at the gene expression level. The quality of the results of qRT‐PCR analysis largely depends on the availability of reliable reference genes (RGs). To date, reliable RGs have not been determined for adaptive evolution studies in insects using a standard approach. Here, we evaluated the reliability of 17 candidate RGs for five Gynaephora populations inhabiting various altitudes of the Tibetan Plateau (TP) using four independent (geNorm, NormFinder, BestKeeper, and the deltaCt method) and one comprehensive (RefFinder) algorithms. Our results showed that EF1‐α, RPS15, and RPS13 were the top three most suitable RGs, and a combination of these three RGs was the most optimal for normalization. Conversely, RPS2,ACT, and RPL27 were the most unstable RGs. The expression profiles of two target genes (HSP70 and HSP90) were used to confirm the reliability of the chosen RGs. Additionally, the expression patterns of four other genes (GPI,HIF1A,HSP20, and USP) associated with adaptation to extreme environments were assessed to explore the adaptive mechanisms of TP Gynaephora species to divergent environments. Each of these six target genes showed discrepant expression patterns among the five populations, suggesting that the observed expression differences may be associated with the local adaptation of Gynaephora to divergent altitudinal environments. This study is a useful resource for studying the adaptive evolution of TP Gynaephora to divergent environments using qRT‐PCR, and it also acts as a guide for selecting suitable RGs for ecological and evolutionary studies in insects.

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Section: Discussionmentioning

confidence: 99%

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Zhang

Wang

et al. 2017

Ecology and Evolution

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“…To evaluate changes in cDNA levels of IFN-γ, IL-12p40, IL-4, IL-13, TNF-α, and IL-6, which are related to asthma induction, RT-PCR analysis was conducted as suggested by Bustin et al [29]. Total RNA was extracted from the lung using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Opuntia humifusamodulates morphological changes characteristic of asthma via IL-4 and IL-13 in an asthma murine model

Lee

Bae

Choi

et al. 2017

Food & Nutrition Research

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Asthma is a chronic pulmonary disease that affects an estimated 235 million people worldwide, but asthma drugs have many adverse effects. Opuntia humifusa (eastern prickly pear) has been used as a food and traditional medicine worldwide; however, its anti-asthmatic effects have not been reported. We evaluated O. humifusa as a potential therapeutic or preventive component of anti-asthmatic drugs. We divided ovalbumin-sensitized mice into the following groups: normal control, asthma-induced control, dexamethasone-treated group (positive control), 50 mg/kg O. humifusa-treated group, 100 mg/kg O. humifusa-treated group, and 500 mg/kg O. humifusa-treated group. Levels of Th1/Th2/Th17-related cytokines were evaluated using RT-PCR, ELISA, and immunohistochemistry. O. humifusa dose-dependently suppressed the morphological changes typically observed in asthma, such as goblet cell hyperplasia, inflammatory cell infiltration, mucous hypersecretion, and relative basement membrane thickening in the respiratory system. These results may be attributable to regulation of Th1-/Th2-/Th17-related factors, especially interleukin (IL)-4 and IL-13. We conclude that O. humifusa is a potential anti-asthmatic functional food. Abbreviations: O. humifusa: Opuntia humifusa; Th: helper T; RT-PCR: real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; WHO: World Health Organization; IFN-γ: interferon gamma; TNF-α: tumor necrosis factor-alpha; IgE: immunoglobulin E; CD: cluster of differentiation; OVA: ovalbumin; DEX: dexamethasone; BALF: bronchoalveolar fluid; H&E: hematoxylin and eosin; PAS: periodic acid-schiff; PBS: phosphate-buffered saline; BM: basement membrane; cDNA: complementary deoxyribonucleic acid; RNA: ribo nucleic acid; RIPA: radioimmunoprecipitation assay; IHC: immunohistochemistry; HPLC: high-performance liquid chromatography; SD: standard deviation; WBC: white blood cells; APCs: antigen-presenting cells

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“…Here we examined the expression of several genes in whole charr embryos, embryonic heads and adult tissues. Primers were designed using the Primer3 tool 68 and checked for self-annealing and heterodimers according to the MIQE guidelines 69 ( S1 Table). Primers for genes with several paralogs were designed for regions conserved among paralogs.…”

Section: Methodsmentioning

confidence: 99%

The developmental transcriptome of contrasting Arctic charr (Salvelinus alpinus) morphs

et al. 2015

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Species and populations with parallel evolution of specific traits can help illuminate how predictable adaptations and divergence are at the molecular and developmental level. Following the last glacial period, dwarfism and specialized bottom feeding morphology evolved rapidly in several landlocked Arctic charr Salvelinus alpinus populations in Iceland. To study the genetic divergence between small benthic morphs and limnetic morphs, we conducted RNA-sequencing charr embryos at four stages in early development. We studied two stocks with contrasting morphologies: the small benthic (SB) charr from Lake Thingvallavatn and Holar aquaculture (AC) charr.The data reveal significant differences in expression of several biological pathways during charr development. There was also an expression difference between SB- and AC-charr in genes involved in energy metabolism and blood coagulation genes. We confirmed differing expression of five genes in whole embryos with qPCR, including lysozyme and natterin-like which was previously identified as a fish-toxin of a lectin family that may be a putative immunopeptide. We also verified differential expression of 7 genes in the developing head that associated consistently with benthic v.s.limnetic morphology (studied in 4 morphs). Comparison of single nucleotide polymorphism (SNP) frequencies reveals extensive genetic differentiation between the SB and AC-charr (~1300 with more than 50% frequency difference). Curiously, three derived alleles in the otherwise conserved 12s and 16s mitochondrial ribosomal RNA genes are found in benthic charr.The data implicate multiple genes and molecular pathways in divergence of small benthic charr and/or the response of aquaculture charr to domestication. Functional, genetic and population genetic studies on more freshwater and anadromous populations are needed to confirm the specific loci and mutations relating to specific ecological traits in Arctic charr.

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The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Cited by 13,152 publications

References 80 publications

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

Opuntia humifusamodulates morphological changes characteristic of asthma via IL-4 and IL-13 in an asthma murine model

The developmental transcriptome of contrasting Arctic charr (Salvelinus alpinus) morphs

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