Determination of the appropriate gene for real-time PCR analysis of the immotalized stromal cell line HS-5 in 2D and 3D culture following melphalan exposure

Kabrah, S; May, J E; Donaldson, C; Morse, H R

Abstract

Although gene expression analysis using real-time quan- titative reverse transcription-polymerase chain reaction (RT-qPCR) is an accurate method to evaluate cell activi- ties and phenotypes, the reliability of the analysis depends on the selection of appropriate reference gene(s) that are highly expressed and stable in experiments (1). Cell be- haviour and gene expression depend on the culture en- vironment where three dimensional (3D) cultures have signi cant impact on cells compared to the traditional two dimensional (2D) (2). In this study, expression and stability of 12 common reference genes were evaluated in the immortalized stromal cell line HS-5 in 2D and 3D (Biomerix, Nano ber and Alvetex) cultures. In addition, gene stability was evaluated in cultures with and without exposure to 32.8 μM melphalan, for one hour. The stabil- ity of the candidate genes were determined by RT-qPCR with analysis by geNorm and NormFinder. The most high- ly expressed genes in normal culture were: 2D (EIF4A2, 35.3 ± 0.2), Biomerix (EIF4A2, 32.1 ± 0.9), Nano ber (ATP5B, 37.05±0.0), and Alvetex (ACTB, 36.641±0.5). However, melphalan-exposed culture was: 2D (CYC1, 38.4 ± 0.7), Biomerix (SDHA, 38.7 ± 0.4), Nano ber (UBC, 36.9 ± 0.0), and Alvetex (β2M, 36.2 ± 0.09). According to the stability value determined by Norm nder the high- est stability value without drug was 2D (EIF4A2, 0.01), Biomerix (CYC1/EIF4A2), Nano ber (YWHAI, 0.055), and Alvetex (YWHAI/18s, 0.206), for the melphalan- exposed culture was 2D (YWHAZ, 0.078), Biomerix (YWHAZ/β2M, 0.01), Nano ber (18s/UBS, 0.123), and Alvetex (18s/UBS/β2M, 0.213). In conclusion, culture technique and treatment have a signi cant impact on reference gene expression. Thus it is essential to analyse the housekeeping genes for each experimental condition to determine the most suitable reference gene to be used within the comparative analysis of toxicity responses.

• Journal Article Type: Article
• Acceptance Date: Jul 15, 2015
• Publication Date: Nov 6, 2015
• Deposit Date: Apr 25, 2017
• Journal: Mutagenesis
• Print ISSN: 0267-8357
• Electronic ISSN: 1464-3804
• Publisher: Oxford University Press (OUP)
• Peer Reviewed: Peer Reviewed
• Volume: 30
• Issue: 6
• Pages: 874
• DOI: https://doi.org/10.1093/mutage/gev074
• Keywords: determination, appropriate gene, real-time PCR analysis, immotalized stromal cell line, HS-5, 2D and 3D culture, melphalan exposure
• Public URL: https://uwe-repository.worktribe.com/output/803533
• Publisher URL: https://academic.oup.com/mutage
• Contract Date: Apr 25, 2017