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Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods:In the present studies we developed qualitycontrol criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A 260 /A 280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix ® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip ® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01).

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Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray , to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg , personnel , reagents , instrumentation , and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories , with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores , and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48 ؎ 3.97) and statistics ( > 0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories. (J Mol

The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.