Selecting suitable reference genes for qPCR normalization: a comprehensive analysis in MCF-7 breast cancer cell line

Abstract 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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Selecting Suitable Reference Genes for qPCR Normalization: A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

10.21203/rs.3.rs-42293/v2 ◽

2020 ◽

Author(s):

Nityanand Jain ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Gene Expression ◽

Cell Line ◽

Reference Gene ◽

Reference Genes ◽

Cancer Cell Line ◽

Internal Controls ◽

Reference Gene Expression ◽

Qpcr Normalization ◽

Suitable Reference ◽

Mcf 7

Abstract BackgroundMCF-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 have 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 devise an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe 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 gene pair while for culture A2, GAPDH-RNA28S was 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-PCBP1-CCSER2 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-PCBP1-CCSER2 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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Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line

Genes ◽

10.3390/genes12101631 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1631

Author(s):

Nityanand Jain ◽

Ingrida Mitre ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina-Dzerve

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Line ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Line ◽

Reference Genes ◽

Cancer Cell Line ◽

Internal Controls ◽

Chain Reactions

Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. Transcriptomic data of 82 HER2-E breast cancer samples from TCGA database were analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from the literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage the use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

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Identification and Validation of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line

10.21203/rs.3.rs-689871/v2 ◽

2021 ◽

Author(s):

Nityanand Jain ◽

Ingrida Mitre ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Line ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Line ◽

Reference Genes ◽

Cancer Cell Line ◽

Internal Controls ◽

Chain Reactions

Abstract Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. Transcriptomic data from 82 HER2-E breast cancer samples from TCGA database was analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

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

10.21203/rs.3.rs-42293/v1 ◽

2020 ◽

Author(s):

Nityanand Jain ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Line ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Reference Gene ◽

Reference Genes ◽

Internal Controls ◽

Expression Behavior ◽

Mcf 7

Abstract 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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Identification and Validation of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line

10.21203/rs.3.rs-689871/v1 ◽

2021 ◽

Author(s):

Nityanand Jain ◽

Ingrida Mitre ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cell Line ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Line ◽

Reference Genes ◽

Good Practice ◽

Cancer Cell Line ◽

Internal Controls

Abstract Background: Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for overcoming and standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use, especially as single internal controls. Hence, it becomes crucial to identify suitable controls. In the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. To identify novel candidate reference genes, we compiled and analyzed transcriptomic data from 82 HER2-E breast cancer samples from TCGA (The Cancer Genome Atlas) database. Twelve genes with relatively stable expression were selected for further analysis. Additionally, 13 RGs from literature were selected for analysis. The expression variations of the candidate genes in SK-BR-3 were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia using Pfaffl’s method.Results: HSP90AB1, DAD1, PFN1, RPL13A and PUM1 were the top five most stable RGs in the SK-BR-3 cell line. However, upon normalization of the GOIs, RPL13A was not a suitable RG candidate. Although geNorm suggests use of two reference genes, as a good practice, we investigated the reference genes in triplets. Accordingly, after normalization of GOIs in hypoxia, we found that the remaining four genes when used in triplets (in any combination) successfully normalized the experimental variations in RT-qPCR experiments.Conclusions: HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

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