Analytical Characteristics of a Noninvasive Gene Expression Assay for Pigmented Skin Lesions

Assay Drug Dev Technol. 2016 Aug;14(6):355-63. doi: 10.1089/adt.2016.724.


We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). This study focuses on analytical characterization of this PLA, including qPCR specificity and sensitivity, optimization of RNA input in qPCR to achieve a desired diagnostic sensitivity and specificity, and analytical performance (repeatability and reproducibility) of this two-gene PLA. All target qPCRs demonstrated a good specificity (100%) and sensitivity (with a limit of detection of 1-2 copies), which allows reliable detection of gene expression changes of LINC and PRAME between melanomas and nonmelanomas. Through normalizing RNA input in qPCR, we converted the traditional gene expression analyses to a binomial detection of gene transcripts (i.e., detected or not detected). By combining the binomial qPCR results of the two genes, an improved diagnostic sensitivity (raised from 52%- 65% to 71% at 1 pg of total RNA input, and to 91% at 3 pg of total RNA input) was achieved. This two-gene PLA demonstrates a high repeatability and reproducibility (coefficient of variation <3%) and all required analytical performance characteristics for the commercial processing of clinical samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Neoplasm / biosynthesis
  • Antigens, Neoplasm / genetics
  • Cell Line, Tumor
  • Gene Expression / genetics*
  • Humans
  • Melanoma / genetics*
  • Melanoma / metabolism
  • Plasmids / biosynthesis
  • Plasmids / genetics
  • RNA, Long Noncoding / biosynthesis
  • RNA, Long Noncoding / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Skin Neoplasms / genetics*
  • Skin Neoplasms / metabolism


  • Antigens, Neoplasm
  • PRAME protein, human
  • RNA, Long Noncoding

Supplementary concepts

  • Melanoma, Cutaneous Malignant