TY - JOUR

T1 - Evaluation of Reference Gene Stability for Investigations of Intracellular Signalling in Human Cancer and Non-Malignant Mesenchymal Stromal Cells

AU - Kosheverova, V.

AU - Schwarz, A.

AU - Kamentseva, R.

AU - Kharchenko, M.

AU - Kornilova, E.

N1 - Export Date: 01 November 2025; Cited By: 2; Correspondence Address: V. Kosheverova; Laboratory of Intracellular Membranes Dynamics, Institute of Cytology, Russian Academy of Sciences, Saint Petersburg, 194064, Russian Federation; email: kosheverova_vera@incras.ru

PY - 2024

Y1 - 2024

N2 - Background: Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins. Methods: The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ∆Ct method) and ranked with the RefFinder web-based tool. Results: We found increased variability in the housekeeping genes’ expression in the cancer cell lines compared to that in normal MSCs. POP4 and GAPDH were identified as the most suitable reference genes in cancer cells, while 18S and B2M were the most suitable in MSCs. POP4 and EIF2B1 were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation. Conclusion: We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression. © 2025 Elsevier B.V., All rights reserved.

AB - Background: Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins. Methods: The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ∆Ct method) and ranked with the RefFinder web-based tool. Results: We found increased variability in the housekeeping genes’ expression in the cancer cell lines compared to that in normal MSCs. POP4 and GAPDH were identified as the most suitable reference genes in cancer cells, while 18S and B2M were the most suitable in MSCs. POP4 and EIF2B1 were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation. Conclusion: We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression. © 2025 Elsevier B.V., All rights reserved.

KW - human cancer cell lines

KW - human mesenchymal stromal cell lines

KW - reference gene stability

KW - RT-qPCR

KW - epidermal growth factor receptor

KW - messenger RNA

KW - A-431 cell line

KW - A-549 cell line

KW - algorithm

KW - Article

KW - comparative study

KW - controlled study

KW - female

KW - gene expression

KW - HeLa cell line

KW - housekeeping gene

KW - human

KW - human cell

KW - intracellular signaling

KW - intracellular transport

KW - MCF-7 cell line

KW - mesenchymal stroma cell

KW - metabolism

KW - post hoc analysis

KW - real time reverse transcription polymerase chain reaction

KW - signal transduction

KW - SK-BR-3 cell line

KW - essential gene

KW - gene expression profiling

KW - gene expression regulation

KW - genetics

KW - mesenchymal stem cell

KW - neoplasm

KW - real time polymerase chain reaction

KW - standard

KW - tumor cell line

KW - Cell Line, Tumor

KW - Gene Expression Profiling

KW - Gene Expression Regulation, Neoplastic

KW - Genes, Essential

KW - Humans

KW - Mesenchymal Stem Cells

KW - Neoplasms

KW - Real-Time Polymerase Chain Reaction

KW - Reference Standards

KW - Signal Transduction

U2 - 10.31083/j.fbs1604026

DO - 10.31083/j.fbs1604026

M3 - статья

VL - 16

JO - Frontiers in Bioscience - Scholar

JF - Frontiers in Bioscience - Scholar

SN - 1945-0516

IS - 4

ER -