A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data

Abstract

BackgroundIn research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.ResultsA number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance.ConclusionEven in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality.

SpxB Regulates O-Acetylation-dependent Resistance of Lactococcus lactis Peptidoglycan to Hydrolysis

“…DNA Microarray Analysis-DNA microarray experiments were performed as described (27,28). Briefly, RNA was isolated from three independent cultures of L. lactis MG1363/pVE3916 (control strain VES4075) and L. lactis MG1363/pspxB ϩ (strain VES3910).…”

“…Briefly, RNA was isolated from three independent cultures of L. lactis MG1363/pVE3916 (control strain VES4075) and L. lactis MG1363/pspxB ϩ (strain VES3910). Slide data were processed and normalized using MicroPrep software (27,28) as described (28). Expression of a gene was considered to be significantly altered when the Cyber-T Bayesian p value was Յ0.001.…”

The Lactococcus lactis CodY Regulon

“…Subsequently, single-strand reverse transcription (amplification) and indirect labeling of 25 g of total RNA, with either Cy3 or Cy5 dye, were performed (including a sample in which the dyes were swapped to correct for dye-specific effects) using the CyScribe post-labeling kit (Amersham Biosciences). Labeled cDNA samples were hybridized to slides representing 2110 open reading frames of L. lactis IL1403 spotted in duplicate and constructed as described (34,35). After overnight hybridization, slides were washed for 1 min at room temperature in 2ϫ SSC, 0.5% SDS and 5 min in 1ϫ SSC, 0.25% SDS to remove nonspecifically hybridized cDNAs.…”

“…Subsequently, individual spot intensities were determined using ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, MD). Slide data were processed and normalized using MicroPrep (35,36), which yielded average ratios of gene expression of mutant over wild type (WT) strain from the data of replicates. Expression of a gene was considered to be significantly altered when its ratio of expression in the mutant compared with the WT was Ͼ1.5 and had a p value Ͻ0.001 and false discovery rate Ͻ0.05 that were determined as described previously (35,37).…”

“…Slide data were processed and normalized using MicroPrep (35,36), which yielded average ratios of gene expression of mutant over wild type (WT) strain from the data of replicates. Expression of a gene was considered to be significantly altered when its ratio of expression in the mutant compared with the WT was Ͼ1.5 and had a p value Ͻ0.001 and false discovery rate Ͻ0.05 that were determined as described previously (35,37). When a significantly up-regulated gene formed part of an operon, other members of that transcriptional unit were included, providing that they showed coregulation.…”