Abstract
Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.
Highlights
HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM Formalin-fixed paraffin-embedded (FFPE) tissue, while the combination of HPRT1 and transferrin receptor (TFRC) genes are the best option in analysing cutaneous squamous cell carcinoma (CSCC) FFPE samples
The aim of the current study is to evaluate the usefulness of ACTB, HPRT1, TATA-box binding protein (TBP) and TFRC
We demonstrated that HPRT1 was the most stable reference gene in CSCC and MM
Summary
The real time PCR is the most accurate and reliable molecular biology technique in gene expression studies for validation of candidate biomarkers [9,10] In these kinds of studies, it is important to consider several subjects: the quality of the sample, the RNA isolation procedure, the stability and degradation of the RNA, the retrotranscription to cDNA, and the qPCR technique [11]. The use of reference genes as internal controls is the most common method for normalizing mRNA data but its use needs to be experimentally validated for specific tissues or cell types and specific experimental designs [13] These issues are still largely ignored, and many reported studies contain poorly normalized qPCR data [14]. The aim of the current study is to evaluate the usefulness of ACTB, HPRT1, TBP and TFRC as candidate reference genes for mRNA expression studies, including CSCC and MM FFPE samples. Establishing the best reference gene will improve the quality of forthcoming expression studies on CSCC or MM samples
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