In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

Background  

Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.     

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.     

Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain

Background  

Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.     

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.     

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages

Background  

Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA).     

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 housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

Background  

Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.     

Exploring valid reference genes for gene expression studies in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>by real-time PCR

Background  

The wild grass species Brachypodium distachyon (Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.     

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.     

An adaptive method for cDNA microarray normalization

Background  

Normalization is a critical step in analysis of gene expression profiles. For dual-labeled arrays, global normalization assumes that the majority of the genes on the array are non-differentially expressed between the two channels and that the number of over-expressed genes approximately equals the number of under-expressed genes. These assumptions can be inappropriate for custom arrays or arrays in which the reference RNA is very different from the experimental samples.     

Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

Background  

Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application.