β-Actin—an unsuitable internal control for RT-PCR

Selvey, Saxon; Thompson, Erik W.; Matthaei, Klaus I.; Lea, Rodney Arthur; Irving, Michael G.; Griffiths, Lyn R.

doi:10.1006/mcpr.2001.0376

Cited by 308 publications

(202 citation statements)

References 12 publications

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“…ACTB was the most appropriate housekeeping gene for cell-free DNA and mRNA quantification (Bronkhorst et al 2016), as well as GAPDH was shown to be a stable gene under heat shock in Ditylum brightwellii (Guo et al 2013). However, other studies suggested that the conventional housekeeping genes were not always highly stably expressed in other animal species or in a complex range of experimental treatments (Schmittgen and Zakrajsek 2000;Selvey et al 2001;Zhu et al 2001;Glare et al 2002;Bas et al 2004). For example, Bas et al (2004) demonstrated that ACTB was not suitable for housekeeping gene using RT-PCR in human T lymphocytes.…”

Section: Discussionmentioning

confidence: 99%

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

Zhang

Gao

Huang

et al. 2017

Italian Journal of Animal Science

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The aim of this study was designed to select housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers. Twelve 3-week-old chickens were randomly selected from 60 yellow-feathered broilers. Then, 12 chickens were killed; the liver and jejunum samples were collected. The gene expression of housekeeping genes (b-actin, ACTB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; hypoxanthine phosphoribosyl transferase 1, HPRT1; ribosomal protein L13, RPL13; TATA box binding protein, TBP; hydroxymethylbilane synthase, HMBS) were determined using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, the expression stabilities of housekeeping genes were analysed using geNorm, Normfinder and BestKeeper programs. The result showed that RPL13 is the most proper gene in liver, GADPH is the most proper gene in jejunum, and HMBS is the most proper gene in all tissues. In conclusion, this result provides the integrated reported evaluation of housekeeping genes for use in expression studies in yellow-feathered broilers. These findings further emphasise the need to accurately validate candidate housekeeping genes in the study before use in gene expression studies using RT-PCR. ARTICLE HISTORY

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Section: Discussionmentioning

confidence: 99%

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

Zhang

Gao

Huang

et al. 2017

Italian Journal of Animal Science

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“…18S rRNA was more reliable than other genes (beta-actin gene, etc.) as an internal control gene for the gene expression research (Mauro and Edelman, 1997;Selvey et al, 2001). So, samples were normalized using the housekeeping gene, 18S rRNA.…”

Section: Rt-pcrmentioning

confidence: 99%

The tissue-specific and developmental expression patterns of the forkhead transcription factor FoxO1 gene in pigs

Liu¹,

Wang²,

Shan³

et al. 2008

J. Anim. Feed Sci.

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The forkhead transcription factor FoxO1 has been studied as the most downstream targets of the signaling pathway in mammals, and played a key role in adipogeneic, cell cycle progression, and energy metabolism and so on. The aim of this study was to investigate the tissue-specifi c and weight-dependent expression patterns of FoxO1 gene in porcine omental adipose tissue and its relation to adipose deposition. Sixty female Duroc × Landrace × Yorkshire pigs in 5 groups, each group containing twelve pigs weighing 1, 20, 40, 60, and 90 kg were used to study developmental expression of FoxO1 gene by means of semi-quantitative RT-PCR. The results showed that FoxO1 mRNA was expressed at a higher level in heart, brain, omental adipose tissue, skeletal muscle, liver and spleen, at a moderate level in kidney, lung, and peritoneal adipose tissue, and at a less level in duodenum, subcutaneous adipose tissue and pancreas. In addition, our data also showed that the expression levels of FoxO1 gene was lowest in the pigs at 20 kg, and continuously increased from 20 to 90 kg body weight (P<0.05). Furthermore, a close positive correlation between the expression levels of FoxO1 gene and the adipose deposition rate was found in pigs (r=0.84, P<0.05).

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“…Variability may also occur when cellular signalling pathways and basic cell metabolism are disturbed (Singh and Green 1993). In addition, the expression of these reference genes may be influenced by experimental conditions (Thellin et al 1999;Selvey et al 2001;Tricarico et al 2002;Arukwe 2006;Valenti et al 2006;Tanic et al 2007), drug and hormone treatments (Bustin 2000;Gorzelniak et al 2001;Huggett et al 2005). Therefore, appropriate reference genes need to be identified and validated for every experimental design.…”

Section: Introductionmentioning

confidence: 99%

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

et al. 2011

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Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C t values. The geNorm analysis showed the following ranking for stability of genes:A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.

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β-Actin—an unsuitable internal control for RT-PCR

Cited by 308 publications

References 12 publications

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

The tissue-specific and developmental expression patterns of the forkhead transcription factor FoxO1 gene in pigs

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

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