A strategy to identify housekeeping genes suitable for analysis in breast cancer diseases

Tilli, Tatiana Martins; Castro, Cláudio da Silva; Tuszyński, Jack A.; Carels, Nicolas

doi:10.1186/s12864-016-2946-1

Cited by 55 publications

(76 citation statements)

References 27 publications

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“…Presumably these adaptations would take place in order to meet the energy demands needed for smoltification and for reorganizing tissues in preparation to life in seawater, which could be related to the higher abundance in smolts of serine-rich coiled-coil domain-containing protein 2, stathmin and fibronectin, all related to cell and microtubule organization [110][111][112]. Curiously, serine-rich coiled-coil domain-containing protein 2 has been proposed as a reliable housekeeping gene in humans but this is unlikely to be the case in rainbow trout given the present results [113].…”

Section: Discussionmentioning

confidence: 62%

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss)

et al. 2020

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The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection. PLOS ONE | https://doi.org/10.1371/journal.pone.0227003 January 3, 2020 1 / 23 OPEN ACCESS Citation: Morro B, Doherty MK, Balseiro P, Handeland SO, MacKenzie S, Sveier H, et al. (2020) Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss). PLoS ONE 15(1): e0227003. https://doi.org/10.

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Section: Discussionmentioning

confidence: 62%

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss)

et al. 2020

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“…CCSER2 is described as a "novel housekeeping gene" (nHKG) by Tilli et al [27], who provided evidence as to its use as a reference gene in breast cancer studies. They demonstrated that expression of CCSER2 is expected to be like the other nHKGs they identi ed that can increase the assessment consistency of normalization.…”

Section: Discussionmentioning

confidence: 99%

“…As pointed out in the MIQE guidelines [24], normalization against a single reference gene is not recommended unless a clear evidence of its uniform expression dynamics is described for the speci c experimental conditions. Many studies have been undertaken previously identifying new stable reference genes over the previous two decades for MCF-7 cell line or breast cancer as whole [25,26,27,28,29,30,31,32].…”

Section: A Widely Accepted Technique For Validation Of Gene Expressiomentioning

confidence: 99%

“…The present study, therefore, aims to ll the void by investigating the gene expression of twelve reference genes that were previously identi ed as stable genes by various studies [25,26,27,28,33,34,35,36,37] for MCF-7 cell line but were not accounted and studied together so as to make an evidence supported recommendation of reference genes to be used for normalization of gene of interest in routinely cultured MCF-7 cell line. Further, the study investigated for any differential reference gene expression in two subclones (culture A1 and A2) cultured over multiple passages (p) to further bolster the selection of appropriate reference genes.…”

Section: A Widely Accepted Technique For Validation Of Gene Expressiomentioning

confidence: 99%

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Which Reference Genes to Choose for qPCR Normalization? A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

Jain¹,

Nitisa²,

Pirsko³

et al. 2020

Preprint

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Background MCF-7 cell line remains the most extensively studied patient derived model in breast cancer research, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, in the past have identified suitable reference genes in various breast cancer cell lines including MCF-7. However, over the course of identification of stable internal controls, a comparative analysis comprising these genes together in a single study have not been previously undertaken. Further, very little is known about how these identified reference genes are expressed in MCF-7 sub-clones and when the cell line is cultured over multiple passages (p), given the heterogenic expression behavior often associated with MCF-7 cell line. We investigated the expression dynamics of 12 previously reported and suggested endogenous reference genes using RT-qPCR, available algorithms (NormFinder, geNorm, BestKeeper etc.) and TCGA transcriptomic analysis within identically cultured two sub-clones (culture A1 and A2) of MCF-7 cell line cultured over multiple passages. Further candidate reference genes were used to normalize 4 genes of interest (2 simulated and 2 backed by qPCR data) to make an evidence-based recommendation of the least variable reference genes that could be used in MCF-7 cell line. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as least variable reference gene pair while for culture A2, GAPDH-RNA28S was recommended. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization with 3 genes and analyzing the combined dataset from culture A1 and A2 (supported by transcriptomic analysis), GAPDH-PCBP1-CCSER2 triplet was found to be least variable and hence potentially more well placed to handle any expression heterogeneity that may arise within sub-clones over multiple passages. Conclusions The variance in expression behavior amongst sub-clones shows the need for exercising caution while selecting and using reference genes for MCF-7. Further, using same reference genes amongst sub-clones can lead to misleading results arising from inaccurate normalization. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls in optimizing intra- and inter-assay gene expression differences.

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“…The internal control gene chosen, therefore, has to be stably expressed and show no difference in expression levels between groups, independent of the tissue type, cell line, experimental conditions, development stage, or disease state [11][12][13] . However, it has been recently shown by many different groups that under certain non-physiological or experimental conditions, the levels of the most commonly used internal control or housekeeping genes in fact change, and are particularly affected in cancer 12,[14][15][16][17][18][19][20] . For example, studies validating housekeeping genes in glioblastoma show that TBP has been the most stable internal control whereas GAPDH shows significant differences between tumour samples and controls 15,19 .…”

Section: Introductionmentioning

confidence: 99%

Analysis of housekeeping genes in the peripheral blood of retinoblastoma patients

Martínez-Sánchez

Hernández-Monge

Moctezuma-Dávila

et al. 2019

Preprint

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Retinoblastoma is a pediatric neoplasia with a high incidence in non-developed countries. Nowadays the diagnosis is clinical and unfortunately in advanced stages of the diseases, which puts the child´s life risk. The search for molecular diagnosis on retinoblastoma is necessary. Due to their location and tendency to migrate, biopsies of retinoblastoma are not recommended then, peripheral blood samples can be a good source for the search of biomarker. RT qPCR is a sensitive method for gene expression quantification; in order to achieve optimal results, a crucial step is the reference gene choice that cannot be neglected under any circumstance. Six of the most commonly used housekeeping genes; GAPDH, HPRT1, B2M, TBP, RPL13a and 18S, were tested in the blood samples of patients diagnosed with retinoblastoma and healthy controls.The HPRT and TBP were found the most reliable genes whereas GAPDH, that is one of the most commonly used genes for normalisation, together with B2M, RPL13a and 18S have to be avoided. Using the selected reference genes, the Rb mRNA showed significant differences between patients and healthy children whereas no differences were found using to control groups. In the present study, we validate blood samples of patients with retinoblastoma.

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A strategy to identify housekeeping genes suitable for analysis in breast cancer diseases

Cited by 55 publications

References 27 publications

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss)

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss)

Which Reference Genes to Choose for qPCR Normalization? A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

Analysis of housekeeping genes in the peripheral blood of retinoblastoma patients

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