Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells

Facci, Marina; Auray, Gaël; Meurens, François; Buchanan, Rachelle; Kessel, Jill van; Gerdts, Volker

doi:10.1016/j.vetimm.2011.01.005

Cited by 29 publications

(22 citation statements)

References 13 publications

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“…Our results identified the combination of SDHA and TBP as a suitable normalizer in T cells as well as in neutrophils. In good agreement, a recent study recommends the use of SDHA as a reference gene in LPS stimulated porcine T cells [27]. Furthermore, TBP has recently been reported to be stably expressed in LPS stimulated monocytes [11].…”

Section: Discussionmentioning

confidence: 72%

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

et al. 2011

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BackgroundThe choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes.ResultsThe mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells.ConclusionsThe study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

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Section: Discussionmentioning

confidence: 72%

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

et al. 2011

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“…Hence, it is possible that an overstimulation of the cells in this study initially caused a shutdown or downregulation in the TLR4 expression and after 6-h exposure to LPS the TLR4 expression was restored back to its original levels (i.e., without the LPS). Although GAPDH was always considered to be the preferred housekeeping gene, recent reports indicate that in some circumstances (including stimulation with LPS), it is more accurate to use a combination of GAPDH with another housekeeping gene than the use of this gene alone (16,35). Nevertheless, we used solely this gene in the interpretations of the RT-PCR results.…”

Section: Discussionmentioning

confidence: 99%

Pulmonary intravascular macrophages as proinflammatory cells in heaves, an asthma-like equine disease

Aharonson-Raz

Lohmann

Townsend

et al. 2012

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…(66,67). Although there are cases when BA can be used as a reference gene (68,69), it is not advisable to use it in situations when a tissue undergoes extensive morphological changes, as expected in different developmental stages, or in rapidly growing tissues, such as cancers.…”

Section: Housekeeping Genes As Reference Genesmentioning

confidence: 99%

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Nestorov¹,

Matić²,

Elaković³

et al. 2013

Journal of Medical Biochemistry

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Summary: Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute Kratak sadr`aj: RT-PCR je prepoznat kao precizna, pouzdana i osetljiva metoda za kvantifikaciju transkripcije gena. Me|utim, ovoj metodi prethodi nekoliko koraka koji predstavljaju kriti~ne ta~ke i izvor potencijalnih gre{aka. Ovi koraci uklju~uju obradu }elijskog materijala, ekstrakciju ĩ uvanje RNK, odre|ivanje koncentracije RNK i sintezu cDNK. Da bi se kompenzovala potencijalna varijabilnost nastala tokom procedure, uvedena je normalizacija ekspresije ciljnih gena. Precizna normalizacija je postala apsolutni preduslov za ta~nu kvantifikaciju ekspresije gena. Postoji nekoliko strategija za normalizaciju eksperimentalnih podataka, uklju~uju}i normalizaciju u odnosu na veli~inu uzorka, ukupnu RNK ili internu kontrolu (referencu). Kao interna (endogena) kontrola naj~e{}e se koriste geni sa stabilnom ekspresijom. Imaju}i u vidu veliku osetljivost, reproducibilnost i ve liki dinami~ki opseg PCR metode, zahtevi za odgova raju}im referentnim genima koji }e se koristiti za normalizaciju podataka postali su veoma restriktivni. Cilj ovog rada je da razjasni koncept normalizacije i prokomentari{e nekoliko statisti~kih algoritama koji su razvijeni kako bi pomogli u validaciji potencijalnih referentnih gena. Pokazuju}i da kori{}enje neodgovaraju}ih referentnih gena (endogenih kontrola) mo`e da dovede do neta~nih rezultata i pogre{ne interpretacije eksperimentalnih podataka, mi se priklju~ujemo tvorcima uputstva MIQE (eng. Minimum Information for Publication of Quantitati...

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Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells

Cited by 29 publications

References 13 publications

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

Pulmonary intravascular macrophages as proinflammatory cells in heaves, an asthma-like equine disease

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

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