Abstract
Real-time quantitative PCR (RT-qPCR) has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference gene selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the optimal reference genes for the relative quantitative analysis of RT-qPCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most frequently used internal reference gene, was significantly unstable between various cell lines. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. These recommended internal reference gene selections may improve the accuracy and reproducibility of RT-qPCR in gene expression analyses of NF1 related tumors.
Highlights
Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome that affects multiple organ systems and has a wide range of variable clinical manifestations, such as pigmentary lesions, skeletal abnormalities [1,2,3]
We investigated the stability of these eleven reference genes in fourteen different NF1 related cell lines, including two non-tumor NF1+/- Schwann cell lines, five benign plexiform neurofibromas (pNF) cell lines and seven malignant malignant peripheral nerve sheath tumors (MPNST) cell lines
It is worth noting that Ubiquitin C (UBC) showed significantly higher Ct values in MPNST samples compared to other samples, suggesting lower mRNA expression level
Summary
Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome that affects multiple organ systems and has a wide range of variable clinical manifestations, such as pigmentary lesions, skeletal abnormalities [1,2,3]. The major defining features in NF1 patients are peripheral nerve sheath tumors, including plexiform neurofibromas (pNF) and malignant peripheral nerve sheath tumors (MPNST) [4]. The majority of NF1 related tumor researches could be categorized into benign tumor studies or malignant tumor studies. Omics studies play an increasingly important role in uncovering disease mechanisms and screening potential therapeutic biomarkers.
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