Development of a new reference standard for microarray experiments

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.

What Is the Best Reference RNA? And Other Questions Regarding the Design and Analysis of Two-color Microarray Experiments

“…Since the reference RNA uses such a large proportion of array resources, and since the reference is the linchpin of the design, selecting an RNA to use as the reference is a choice that investigators take very seriously. Some previous work has discussed choices for reference RNAs (Gorreta, F. et al, 2004;He, X. R. et al, 2004;Novoradovskaya, N. et al, 2004;Yang, I. V. et al, 2002). Each of these papers asserts that the most important quality of a reference RNA is that it has good representation of all of the genes on the array, i.e.…”

Identification of thioredoxin reductase 1-regulated genes using small interference RNA and cDNA microarray

“…A microarray (named "Human_1_20K_Chip") was constructed as previously described 13 from approximately 20,000 human cDNA clones purchased from Research Genetics (Huntsville, AL, USA). The list of genes is available at http://www.gmu.edu/centers/genomics/ research/keys.html.…”

“…The analysis of the direct hybridization of two RNA populations was performed as previously described. 13 Briefly, the local background value, as provided by QuantArray software, was subtracted from the intensity level of each gene in each channel. A filtering step was performed in each channel separately, using thresholds equal to µ NC + 1σ NC (with µ NC , and σ NC denoting the mean and standard deviation of the background-corrected intensity of the negative controls printed on the slide).…”

Hepatic Stellate Cell and Myofibroblast-Like Cell Gene Expression in the Explanted Cirrhotic Livers of Patients Undergoing Liver Transplantation

“…Aminoallyl-labeled cDNA samples were coupled with Cy5 fluorescent dye. Prehybridization and hybridization of microarray slides were performed according to a standard procedure [11]. Each sample, which had been derived from a particular fraction from an individual patient (i.e., not pooled), was independently hybridized in triplicate.…”