Selection of internal references for RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

PLoS One. 2021 Feb 25;16(2):e0241821. doi: 10.1371/journal.pone.0241821. eCollection 2021.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Line, Tumor
  • Genes, Neurofibromatosis 1*
  • Humans
  • Neurofibromatosis 1 / genetics*
  • Neurofibromin 1 / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Schwann Cells / metabolism*

Substances

  • NF1 protein, human
  • Neurofibromin 1

Grants and funding

ZC W, QC201803, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, http://www.9hospital.com.cn/djy_web/html/djy/portal/index/index.htm; QF L, YBKA201901, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, http://www.9hospital.com.cn/djy_web/html/djy/portal/index/index.htm; ZC W, 201809004, Shanghai Municipal People’s Government, http://english.shanghai.gov.cn/nw46669/index.html; ZC W, 19CG18, Shanghai Education Development Foundation and Shanghai Municipal Education Commission, http://www.shedf.org.cn/, http://edu.sh.gov.cn/; QF L, 19JC1413, Science and Technology Commission of Shanghai Municipality, http://stcsm.sh.gov.cn/; ZC W, 20QA1405600, Science and Technology Commission of Shanghai Municipality, http://stcsm.sh.gov.cn/; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.