Development of a new reference standard for microarray experiments

Biotechniques. 2004 Jun;36(6):1002-9. doi: 10.2144/04366RR01.

Abstract

Often microarray studies require a reference to indirectly compare the samples under observation. References based on pooled RNA from different cell lines have already been described (here referred to as RNA-R), but they usually do not exhaustively represent the set of genes printed on a chip, thus requiring many adjustments during the analyses. A reference could also be generated in vitro transcribing the collection of cDNA clones printed on the microarray in use (here referred to as T3-R). Here we describe an alternative and simpler PCR-based methodology to construct a similar reference (Chip-R), and we extensively test and compare it to both RNA-R and T3-R. The use of both Chip-R and T3-R dramatically increases the number of signals on the slides and gives more reproducible results than RNA-R. Each reference preparation is also evaluated in a simple microarray experiment comparing two different RNA populations. Our results show that the introduction of a reference always interferes with the analysis. Indeed, the direct comparison is able to identify more up- or down-regulated genes than any reference-mediated analysis. However, if a reference has to be used, Chip-R and T3-R are able to guarantee more reliable results than RNA-R.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Calibration / standards
  • DNA / analysis*
  • DNA / standards*
  • Gene Expression Profiling / methods*
  • Gene Expression Profiling / standards*
  • Guidelines as Topic
  • Internationality
  • Oligonucleotide Array Sequence Analysis / instrumentation
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotide Array Sequence Analysis / standards*
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • DNA