Selection of optimal reference genes for normalization in quantitative RT-PCR

Background  

Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.     

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Background  

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.     

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30?°C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14?+?U6 for SE tissues and snoR14?+?U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14?+?U6, snoR14?+?U5 were identified.     

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results     

Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Selection of reference genes for normalisation of real-time RT-PCR in brain-stem death injury in Ovis aries

Background  

Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD) is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD.     

蜜环菌(Armillaria mellea)内参基因的筛选

[背景]蜜环菌属(Armillaria)是一类营腐生或寄生生活的药食两用型真菌,研究其功能基因表达具有重要意义。[目的]筛选并获得蜜环菌(Armillaria mellea)最稳定的内参基因。[方法]以蜜环菌(A.mellea)541为研究对象,以马铃薯琼脂糖培养基培养的菌丝和菌索为对照组,以添加还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶抑制剂氯化二亚苯基碘鎓(diphenyleneiodonium chloride,DPI)培养的菌丝和菌索为抑制剂组,以添加木屑培养的菌丝和菌索为诱导剂组,利用RT-qPCR技术和BestKeeper程序系统评估候选内参基因ACT-1、α-TUB、β-TUB 1、γ-TUB、UBQ、EF-lγ、18S rRNA biogenesis protein基因(18S rRNA BP)和GAPDH的表达量稳定性。[结果]内参基因EF-1γ在对照组、DPI抑制剂组和木屑诱导剂组中的表达量稳定性最好。[结论]EF-1γ为蜜环菌(A.mellea)的最佳内参基因,为蜜环菌属真菌功能基因表达研究提供了参考。     

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman^® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.     

Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR

Background  

Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared.     

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

Key message

The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.

Abstract

Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.