Selection of reliable reference genes for gene expression studies in peach using real-time PCR

Background  

RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.     

Selection of reference genes for gene expression studies in human neutrophils by real-time PCR

Background  

Reference genes, which are often referred to housekeeping genes, are frequently used to normalize 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 reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils.     

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.     

An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

Background  

Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents.     

Selection of reference genes for expression studies with fish myogenic cell cultures

Background  

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.     

Reference gene alternatives to <Emphasis Type="Italic">Gapdh</Emphasis> in rodent and human heart failure gene expression studies

Background  

Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.     

Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans

Background  

Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data.     

Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar

Background  

RT-qPCR is a powerful tool for analysing gene expression. It depends on measuring the increase in fluorescence emitted by a DNA-specific dye during the PCR reaction. For relative quantification, where the expression of a target gene is measured in relation to one or multiple reference genes, various mathematical approaches are published. The results of relative quantification can be considerably influenced by the chosen method.     

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.     

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.