A unified censored normal regression model for qPCR differential gene expression analysis

PLoS One. 2017 Aug 17;12(8):e0182832. doi: 10.1371/journal.pone.0182832. eCollection 2017.

Abstract

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. Prior to downstream statistical analysis, RT-qPCR fluorescence amplification curves are summarized into one single value, the quantification cycle (Cq). When RT-qPCR does not reach the limit of detection, the Cq is labeled as "undetermined". Current state of the art qPCR data analysis pipelines acknowledge the importance of normalization for removing non-biological sample to sample variation in the Cq values. However, their strategies for handling undetermined Cq values are very ad hoc. We show that popular methods for handling undetermined values can have a severe impact on the downstream differential expression analysis. They introduce a considerable bias and suffer from a lower precision. We propose a novel method that unites preprocessing and differential expression analysis in a single statistical model that provides a rigorous way for handling undetermined Cq values. We compare our method with existing approaches in a simulation study and on published microRNA and mRNA gene expression datasets. We show that our method outperforms traditional RT-qPCR differential expression analysis pipelines in the presence of undetermined values, both in terms of accuracy and precision.

MeSH terms

  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism
  • Child
  • Child, Preschool
  • Gene Expression Profiling / methods*
  • Gene Expression Profiling / standards
  • Humans
  • MicroRNAs / genetics
  • Molecular Diagnostic Techniques / methods*
  • Molecular Diagnostic Techniques / standards
  • N-Myc Proto-Oncogene Protein / genetics
  • N-Myc Proto-Oncogene Protein / metabolism
  • Neuroblastoma / diagnosis
  • Neuroblastoma / genetics*
  • Neuroblastoma / metabolism
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Reference Standards
  • Sensitivity and Specificity

Substances

  • Biomarkers, Tumor
  • MYCN protein, human
  • MicroRNAs
  • N-Myc Proto-Oncogene Protein

Grants and funding

This study was sponsored by a UGent Special Research Fund Concerted Research Actions (GOA grant number BOF16-GOA-023). Biogazelle provided support in the form of salaries for authors JH and JV, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.