Development of a new reference standard for microarray experiments

• Published in: BioTechniques Vol. 36; no. 6; pp. 1002 - 1009
• Main Authors: Gorreta, Francesco, Barzaghi, Dagania, VanMeter, Amy J, Chandhoke, Vikas, Giacco, Luca Del
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.06.2004; Eaton; Taylor & Francis Group
• Subjects: Biological and medical sciences; Calibration - standards; Diverse techniques; DNA - analysis; DNA - standards; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling - methods; Gene Expression Profiling - standards; Guidelines as Topic; Internationality; Molecular and cellular biology; Oligonucleotide Array Sequence Analysis - instrumentation; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide Array Sequence Analysis - standards; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Development; Standard; Reference; Standards; DNA chip
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/04366RR01

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.