Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

Boryana Stamova, Michelle L Apperson, Wynn L. Walker, Yingfang Tian, Huichun Xu, Peter Adamczy, Xinhua Zhan, Da Liu, Bradley Ander, Isaac H. Liao, Jeffrey Gregg, Renee J. Turner, Glen Jickling, Lisa Lit, Frank R Sharp

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Background: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods. Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results. Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion. The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Original languageEnglish (US)
Article number49
JournalBMC Medical Genomics
Volume2
DOIs
StatePublished - 2009

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Gene Expression
Genes
Tourette Syndrome
Polymerase Chain Reaction
Muscular Dystrophies
Human Genome
Autistic Disorder
Migraine Disorders
Stroke
RNA

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics

Cite this

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood. / Stamova, Boryana; Apperson, Michelle L; Walker, Wynn L.; Tian, Yingfang; Xu, Huichun; Adamczy, Peter; Zhan, Xinhua; Liu, Da; Ander, Bradley; Liao, Isaac H.; Gregg, Jeffrey; Turner, Renee J.; Jickling, Glen; Lit, Lisa; Sharp, Frank R.

In: BMC Medical Genomics, Vol. 2, 49, 2009.

Research output: Contribution to journalArticle

Stamova, Boryana ; Apperson, Michelle L ; Walker, Wynn L. ; Tian, Yingfang ; Xu, Huichun ; Adamczy, Peter ; Zhan, Xinhua ; Liu, Da ; Ander, Bradley ; Liao, Isaac H. ; Gregg, Jeffrey ; Turner, Renee J. ; Jickling, Glen ; Lit, Lisa ; Sharp, Frank R. / Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood. In: BMC Medical Genomics. 2009 ; Vol. 2.
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abstract = "Background: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods. Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results. Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion. The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.",
author = "Boryana Stamova and Apperson, {Michelle L} and Walker, {Wynn L.} and Yingfang Tian and Huichun Xu and Peter Adamczy and Xinhua Zhan and Da Liu and Bradley Ander and Liao, {Isaac H.} and Jeffrey Gregg and Turner, {Renee J.} and Glen Jickling and Lisa Lit and Sharp, {Frank R}",
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AU - Stamova, Boryana

AU - Apperson, Michelle L

AU - Walker, Wynn L.

AU - Tian, Yingfang

AU - Xu, Huichun

AU - Adamczy, Peter

AU - Zhan, Xinhua

AU - Liu, Da

AU - Ander, Bradley

AU - Liao, Isaac H.

AU - Gregg, Jeffrey

AU - Turner, Renee J.

AU - Jickling, Glen

AU - Lit, Lisa

AU - Sharp, Frank R

PY - 2009

Y1 - 2009

N2 - Background: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods. Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results. Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion. The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

AB - Background: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods. Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results. Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion. The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

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