Dataset Information

Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies.

ABSTRACT: Five potential reference genes for RT-qPCR application, namely histone H3, beta-actin, GAPDH, ubiquitin and 18S rRNA, were evaluated for normalization of gene expression in four selected tissues (liver, kidney, thyroid and abdominal fat). Tissues were derived from fattening pigs exposed to different amounts and type of dietary iodine. Two software applications (geNorm and NormFinder) were used to evaluate the stability of the potential reference genes. All studied genes displayed high expression stability but different stability patterns between the investigated tissues. The results suggest GAPDH and 18S rRNA as reference genes applicable in all tissues investigated. Beta-actin and histone H3 are suitable reference genes for all tissues investigated except fat. In contrast, ubiquitin should be excluded from use as a reference gene in the porcine tissues analyzed due to variations in expression levels, despite the good expression stability.

SUBMITTER: Li Q

PROVIDER: S-EPMC3111629 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies.

Li Qimeng Q Domig Konrad Johann KJ Ettle Thomas T Windisch Wilhelm W Mair Christiane C Schedle Karl K

International journal of molecular sciences 20110307 3

Five potential reference genes for RT-qPCR application, namely histone H3, beta-actin, GAPDH, ubiquitin and 18S rRNA, were evaluated for normalization of gene expression in four selected tissues (liver, kidney, thyroid and abdominal fat). Tissues were derived from fattening pigs exposed to different amounts and type of dietary iodine. Two software applications (geNorm and NormFinder) were used to evaluate the stability of the potential reference genes. All studied genes displayed high expression ...[more]

PMID: 21673918

Similar Datasets

Project description:BackgroundRT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability.ResultsUsing the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most 'stable' genes and that gene 'stability' is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes.ConclusionTo minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated "housekeeping" genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.

Dataset Information

Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies.

Publications

Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets