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Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.
Fernanda Wisnieski1, Danielle Queiroz Calcagno, Mariana Ferreira Leal
1Fernanda Wisnieski, Danielle Queiroz Calcagno, Mariana Ferreira Leal, Leonardo Caires dos Santos, Carolina de Oliveira Gigek, Elizabeth Suchi Chen, Thaís Brilhante Pontes, Marília de Arruda Cardoso Smith, Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo 04023900, Brazil.
Identifying optimal reference genes is crucial for accurate gene expression studies in gastric cancer. ACTB + B2M is recommended for cell lines, while GAPDH + B2M or ACTB + B2M is best for gastric tissues.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Accurate gene expression analysis relies on stable reference genes for normalization.
- Gastric cancer research requires validated reference genes for reliable quantitative real-time PCR (RT-qPCR) results.
- Previous studies have not comprehensively evaluated reference gene suitability in diverse gastric sample types.
Purpose of the Study:
- To identify the most suitable reference genes for gene expression studies in gastric adenocarcinoma tissues and cell lines.
- To determine the optimal single and combination reference genes for normalization using multiple analytical software.
- To validate the findings by assessing the expression of a target gene, DNMT1, under different normalization strategies.
Main Methods:
- Suitability of ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed using RT-qPCR in gastric tissues and cell lines.
- NormFinder, geNorm™, BestKeeper, and DataAssist™ were employed to rank reference genes.
- GenEx software determined the optimal number of reference genes, and DNMT1 expression was quantified for validation.
Main Results:
- ACTB emerged as the best single reference gene across all sample types.
- GAPDH + B2M or ACTB + B2M proved optimal for gastric tissues, while ACTB + B2M was best for cell lines.
- Two or three reference genes were deemed optimal for gastric tissues; DNMT1 expression showed similar patterns regardless of normalization method.
Conclusions:
- ACTB + B2M is the preferred reference gene combination for gastric cell line studies.
- GAPDH + B2M or ACTB + B2M is recommended for normalizing gene expression in all gastric tissues.
- These findings provide essential guidance for robust gene expression analysis in gastric cancer research.
