Evaluation of Reference Genes in Glenea cantor (Fabricius) by Using qRT-PCR

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Abstract

Kapok is the main host of Glenea cantor (Fabricius), which causes serious damage and is difficult to control. In severe cases, it often causes the kapok trees to die continuously, which seriously affects the results of urban landscaping. To provide reference for the functional research on related genes in G. cantor, we screened the stable expression of candidate reference genes at different developmental stages (i.e., eggs, larvae, pupae, and adults), in various adult tissues (i.e., head, thorax, abdomen, feet, antennae, and wings), and sexes (i.e., male pupae, female pupae, male adults, and female adults). In this study, 12 candidate reference genes (i.e., ACTINLIKE, ACTININ, TUB, RPL36, RPL32, RPS20, TBP, GAPDH, 18S rRNA, EF1A1, EF1A2, and UBQ) were evaluated using different adult tissues, developmental stages, and sexes. RefFinder, geNorm, NormFinder, and BestKeeper were used to evaluate and comprehensively analyze the stability of the expression of the candidate reference genes. The results show that RPL32 and EF1A1 were the most suitable reference genes in the different adult tissues, and RPL36 and EF1A1 were best at the different developmental stages. RPL36 and EF1A2 were the best fit for the qRT-PCR reference genes in the different sexes, while RPL36 and EF1A1 were the most appropriate qRT-PCR reference genes in all samples. Results from geNorm showed that the optimal number of reference genes was two. We also surveyed the expression of cellulase at the different developmental stages and in the different adult tissues. Results further verified the reliability of the reference genes, and confirmed the best reference genes under the different experimental conditions. This study provides a useful tool for molecular biological studies on G. cantor.

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The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson … (2009)

Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

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Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.

Claus Andersen, Jens Ledet Jensen, Torben Ørntoft (2004)

Accurate normalization is an absolute prerequisite for correct measurement of gene expression. For quantitative real-time reverse transcription-PCR (RT-PCR), the most commonly used normalization strategy involves standardization to a single constitutively expressed control gene. However, in recent years, it has become clear that no single gene is constitutively expressed in all cell types and under all experimental conditions, implying that the expression stability of the intended control gene has to be verified before each experiment. We outline a novel, innovative, and robust strategy to identify stably expressed genes among a set of candidate normalization genes. The strategy is rooted in a mathematical model of gene expression that enables estimation not only of the overall variation of the candidate normalization genes but also of the variation between sample subgroups of the sample set. Notably, the strategy provides a direct measure for the estimated expression variation, enabling the user to evaluate the systematic error introduced when using the gene. In a side-by-side comparison with a previously published strategy, our model-based approach performed in a more robust manner and showed less sensitivity toward coregulation of the candidate normalization genes. We used the model-based strategy to identify genes suited to normalize quantitative RT-PCR data from colon cancer and bladder cancer. These genes are UBC, GAPD, and TPT1 for the colon and HSPCB, TEGT, and ATP5B for the bladder. The presented strategy can be applied to evaluate the suitability of any normalization gene candidate in any kind of experimental design and should allow more reliable normalization of RT-PCR data.

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Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Nicholas Silver, Steve Best, Jie Jiang … (2006)

Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.