Identification of artifactual microarray probe signals constantly present in multiple sample types

Biotechniques. 2012 Aug;53(2):91-8. doi: 10.2144/0000113903.

Abstract

The detection, identification, and quantitation of transcripts have evolved from simple Northern analysis, cDNA cloning, and sequencing to RT-PCR, microarrays, and now digital gene expression using ultra-high-throughput RNA sequencing (RNA-Seq). During the course of our studies we observed that some microarray probes show very high signal intensity values yet are discordant when compared with RNA-Seq. A total of 99 probes from approximately 30,000 were identified as consistently discordant in four human tissues or cell lines. Interestingly, this set of discordant probes appears array-dependent. Among the 99 probes identified, 70 constantly exhibited a high signal in all 713 available samples surveyed using the Illumina HumanHT-12v4 platform. Some were discordant with additional probes that annotated the same genes. Absence of a number of these transcripts was confirmed by quantitative RT-PCR (qRT-PCR). Our findings suggest that one must be cautious, as some array probes do not capture the level of the target.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • DNA, Complementary / genetics
  • Female
  • Gene Expression Profiling / methods*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Male
  • Nucleic Acid Probes / genetics
  • Oligonucleotide Array Sequence Analysis / methods*
  • Placenta / metabolism
  • Pregnancy
  • RNA / genetics*
  • RNA / isolation & purification
  • Real-Time Polymerase Chain Reaction / methods
  • Skin / cytology
  • Spermatozoa / metabolism
  • Testis / metabolism

Substances

  • DNA, Complementary
  • Nucleic Acid Probes
  • RNA