Development of a new reference standard for microarray experiments.
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.
Gorreta
Francesco
F
George Mason University, Manassas, VA, USA. fgorret1@gmu.edu
Barzaghi
Dagania
D
VanMeter
Amy J
AJ
Chandhoke
Vikas
V
Del Giacco
Luca
L
D002138:Calibration / Q000592:standards; D004247:DNA / Q000032:analysis* / Q000592:standards*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D017408:Guidelines as Topic; D038622:Internationality; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA