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

ABSTRACT: The selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data. Here we outline a strategy to identify housekeeping genes that could serve as suitable internal control for comparative analyses of gene expression data in breast cancer cell lines and tissues obtained by high throughput sequencing and quantitative real-time PCR (qRT-PCR).The strategy proposed includes the large-scale screening of potential candidate reference genes from RNA-seq data as well as their validation by qRT-PCR, and careful examination of reference data from the International Cancer Genome Consortium, The Cancer Genome Atlas and Gene Expression Omnibus repositories.The identified set of reference genes, also called novel housekeeping genes that includes CCSER2, SYMPK, ANKRD17 and PUM1, proved to be less variable and thus potentially more accurate for research and clinical analyses of breast cell lines and tissue samples compared to the traditional housekeeping genes used to this end.These results highlight the importance of a massive evaluation of housekeeping genes for their relevance as internal control for optimized intra- and inter-assay comparison of gene expression.We developed a strategy to identify and evaluate the significance of housekeeping genes as internal control for the intra- and inter-assay comparison of gene expression in breast cancer that could be applied to other tumor types and diseases.

SUBMITTER: Tilli TM

PROVIDER: S-EPMC4986254 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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

Tilli Tatiana M TM Tilli Tatiana M TM Castro Cláudio da Silva Cda S Tuszynski Jack A JA Carels Nicolas N

BMC genomics 20160815 1

<h4>Background</h4>The selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data. Here we outline a strategy to identify housekeeping genes that could serve as suitable internal control for comparative analyses of gene expression data in breast cancer cell lines and tissues obtained by high throughput sequencing and quantitative real-time PCR (qRT-PCR).<h4>Methods</h4>The strategy proposed includes the large-scale screening of potential candidate ref ...[more]

PMID: 27526934

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Project description:BackgroundHypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative[Formula: see text], RefFinder, and the Livak method.ResultsGene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions.ConclusionsWe have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

Project description:BACKGROUND:RNA-seq is poised to play a major role in the management of kidney transplant patients. Rigorous definition of housekeeping genes (HKG) is essential for further progress in this field. Using single genes or a limited set HKG is inherently problematic since their expression might be altered by specific diseases in the patients being studied. METHODS:To generate a HKG set specific for kidney transplantation, we performed RNA-sequencing from renal allograft biopsies collected in a variety of clinical settings. Various normalization methods were applied to identify transcripts that had a coefficient of variation of expression that was below the 2nd percentile across all samples, and the corresponding genes were designated as housekeeping genes. Comparison with transcriptomic data from the Gene Expression Omnibus (GEO) database, pathway analysis and molecular biological functions were utilized to validate the housekeeping genes set. RESULTS:We have developed a bioinformatics solution to this problem by using nine different normalization methods to derive large HKG gene sets from a RNA-seq data set of 47,611 transcripts derived from 30 biopsies. These biopsies were collected in a variety of clinical settings, including normal function, acute rejection, interstitial nephritis, interstitial fibrosis/tubular atrophy and polyomavirus nephropathy. Transcripts with coefficient of variation below the 2nd percentile were designated as HKG, and validated by showing their virtual absence in diseased allograft derived transcriptomic data sets available in the GEO. Pathway analysis indicated a role for these genes in maintenance of cell morphology, pyrimidine metabolism, and intracellular protein signaling. CONCLUSIONS:Utilization of these objectively defined HKG data sets will guard against errors resulting from focusing on individual genes like 18S RNA, actin & tubulin, which do not maintain constant expression across the known spectrum of renal allograft pathology.

Project description:BACKGROUND:MCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study has not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to postulate an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies. 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 the least variable reference genes while for culture A2, GAPDH-RNA28S were identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-CCSER2-PCBP1 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress. CONCLUSIONS:The variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-CCSER2-PCBP1 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

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

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

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