The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization

Dheda, Keertan; Huggett, Jim F.; Chang, Jun; Kim, L.U.; Bustin, Stephen A.; Johnson, Margaret; Rook, G.A.W.; Zumla, Alimuddin

doi:10.1016/j.ab.2005.05.022

Cited by 566 publications

(447 citation statements)

References 8 publications

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“…What is remarkable is that both geNorm and NormFinder rank HMBS as well as the traditional reference genes GAPDH and 18S as most unstable genes. Several other studies also described that GAPDH as well as other traditional reference genes are not suitable as internal reference genes (Dheda et al, 2004;Dheda et al, 2005). Therefore, we strongly advise to check the expression stability of these genes before using them for normalization purposes.…”

Section: Discussionmentioning

confidence: 99%

“…They are supposed to have stable expression levels in different cell types and tissues, across developmental stages and various conditions. Several studies, however, have demonstrated that expression levels of traditional HKGs can vary under different experimental conditions (Deindl et al, 2002;Dheda et al, 2005;Radonic et al, 2004). In addition, normalization to a single control gene can lead to erroneous normalization.…”

Section: Introductionmentioning

confidence: 99%

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Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Nelissen

Smeets

Mulder

et al. 2010

Journal of Neuroscience Methods

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Quantitative real time polymerase chain reaction (qPCR) has become a widely used tool to examine gene expression levels. Reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple reference genes is becoming the standard, although the most suitable reference genes depend on the applied treatment as well as the tissue or cell type studied. In this study the stability of various reference genes was investigated in cultures of oligodendrocytes derived from either mature or neonatal rats, the latter also in the presence of the liver X receptor (LXR) agonist.The expression stability of ten commonly used reference genes (HPRT, GAPDH, 18S, ActB, CycA, Tbp, Rpl13A, YWHAZ, HMBS, Pgk1) was analyzed using geNorm and NormFinder.When comparing the different types of cell cultures, Rpl13A, CycA, Pgk1 and YWHAZ were identified as most stable genes. After LXR agonist treatment, CycA, Pgk1 and Rpl13A were found to be the most stable by both geNorm and NormFinder. HMBS and the commonly used housekeeping genes GAPDH and 18S turned out to be the most variable according to geNorm and NormFinder. In conclusion, the use of multiple reference genes, instead of only one, in qPCR experiments with rat oligodendrocytes is strongly advised and standard housekeeping genes such as GAPDH and 18S are not recommended as they appear to be relatively unstable under the experimental conditions used. Reference gene selection should always be performed for each individual experiment, since useful reference genes are very specific for every situation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Nelissen

Smeets

Mulder

et al. 2010

Journal of Neuroscience Methods

View full text Add to dashboard Cite

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“…Reference genes most frequently are used as internal standards and are selected on their supposedly equal expression in each cell of a specific tissue regardless of the individual. In human biomedical research, there are several reports on evaluating reference genes [9][10][11] or the qPCR technique [12][13][14]. To our knowledge, the evaluation of reference genes for accurate normalization of gene expression has not been done extensively for domestic animals [15][16][17].…”

Section: Pcr (Qpcr)mentioning

confidence: 99%

Development and evaluation of canine reference genes for accurate quantification of gene expression

Brinkhof

Spee

Rothuizen

et al. 2006

Analytical Biochemistry

223

178

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In determining relative gene expression by quantitative measurements of mRNA levels using real-time quantitative PCR, internal standards such as reference genes are essential. Large-scale studies evaluating (candidate) reference genes for veterinary research have not been conducted as thoroughly as for human research, although they are equally important. Our goal was to design and evaluate a genome-wide panel of reference genes from different functional classes. First, primers were optimized using mRNA from canine cell lines and from 30 tissues of one dog as template and SYBR green as fluorescent probe. Second, the expression variation and stability of a gene within one specific tissue were determined. Prostate, kidney, mammary gland, left ventricle, and liver tissues from five to nine dogs of different breeds, sexes, ages, body weights, and disease status were used. Averaging relative stabilities over these tissues revealed the usefulness of individual genes as reference genes. Furthermore, according to expression variation of a reference gene within a specific tissue, usually two to four reference genes are sufficient. Taken together, ribosomal protein S19 (RPS19), ribosomal protein S5 (RPS5), b-2-microglobulin (B2M), and hypoxanthine phosphoribosyltransferase (HPRT) are advocated. However, the optimal set of reference genes depends on the tissue and should be selected and evaluated for each series of experiments.

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“…However, it is essential to validate reference genes for each bacterial species and each specific experiment to be able to control for nonbiological variation and thereby obtain accurate and reliable gene expression data. Selection of unstable, unvalidated reference genes can result in miscalculations of gene expression levels and lead to incorrect conclusions (6). Several publications have underlined the importance of using multiple reference genes, as no single, universal reference gene exists.…”

Section: Discussionmentioning

confidence: 99%

Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

Lent

Vanrompay

2016

Journal of Veterinary Research

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Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

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The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization

Cited by 566 publications

References 8 publications

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Development and evaluation of canine reference genes for accurate quantification of gene expression

Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

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