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Abstract: Data preprocessing including proper normalization and adequate quality control before complex data mining is crucial for studies using the cDNA microarray technology. We have developed a simple procedure that integrates data filtering and normalization with quantitative quality control of microarray experiments. Previously we have shown that data variability in a microarray experiment can be very well captured by a quality score qcom that is defined for every spot, and the ratio distribution depends on qcom. Utilizing this knowledge, our data-filtering scheme allows the investigator to decide on the filtering stringency according to desired data variability, and our normalization procedure corrects the qcom-dependent dye biases in terms of both the location and the spread of the ratio distribution. In addition, we propose a statistical model for false positive rate determination based on the design and the quality of a microarray experiment. The model predicts that a lower limit of 0.5 for the replicate concordance rate is needed in order to be certain of true positives. Our work demonstrates the importance and advantages of having a quantitative quality control scheme for microarrays. Contact: xujing@mcw.edu * To whom correspondence should be addressed.

Background: The development of vascular disease involves the interaction of genetic and environmental factors. Because vascular disease is a major contributor to mortality in Western societies, we hypothesized that deleterious polymorphisms associated with hemostasis decrease in frequency among a healthy population as a function of age.Methods: The frequencies of factor V G1691A Leiden (FVL), factor II (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, glycoprotein Ia (GPIa) C807T, glycoprotein IIIa (PlA1/PlA2) T1565C, and angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) alleles were determined among 2689 healthy Caucasian whole-blood donors. For analysis, participants were divided into three age groups: 17–39 years (n = 979; 505 males and 474 females), 40–59 years (n = 900; 526 males and 374 females), and 60–85 years (n = 810; 530 males and 280 females).Results: The PlA2 allele frequency decreased from 17.5% to 15.7% and 14.1% in the 17–39 years, 40–59 years, and 60–85 years age groups, respectively (n = 5094 alleles; P = 0.025). Among ACE DD males, the PlA2 allele frequency decreased from 20.8% to 16.1% and 9.1% in the same groups, respectively (n = 810 alleles; P = 0.001). No statistically significant decrease in genotype or allele frequency was observed among carriers of FVL, FII 20210A, MTHFR 677T, GPIa 807T, or ACE D.Conclusions: These data suggest that PlA2 carriers, especially those who are ACE DD, are statistically less prevalent among older healthy blood donors compared with their younger counterparts. These observations suggest an important, deleterious, time-dependent impact of the PlA2 allele, as well as the ACE DD/PlA2 allelic combination, on overall health and longevity.

Background: The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis. Methods: Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the InvaderTM assay, utilizes a 5′ “invading” oligonucleotide and a partially overlapping 3′ “signal” oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5′ flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5′ end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio. Results: Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR. Conclusion: We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.

The factor II (prothrombin) G20210A mutation (PT20210) is a recognized genetic risk factor for increased plasma prothrombin and venous thrombosis that is present in 6–7% of thrombosis patients and 1–2% of healthy individuals (1)(2)(3). The first occurrence of deep venous thrombosis is increased 1.4- to 5.6-fold in individuals carrying at least one mutant allele compared with the incidence in individuals without this mutation (4)(5). In this report, we describe the novel application of the Factor II Invader® assay for the genotyping of patient specimens for the prothrombin G20210A mutation. The Invader assay is a sensitive, homogeneous, DNA-probe-based system for quantitatively detecting specific mutations directly from genomic DNA (6)(7)(8) as described previously by Hessner et al. (9) and Ledford et al. (10) To evaluate the suitability of the Invader assay for detection of the prothrombin mutation, peripheral blood specimens from 528 patients (from four different sites) were collected in EDTA or sodium citrate tubes and genotyped. The four participating sites were ( a ) the University of Wisconsin Hospital and Clinics Molecular Pathology Laboratory (Madison, WI), ( b ) the Blood Center of Southeastern Wisconsin (Milwaukee, WI), ( c ) the University of Miami, Department of Pathology (Miami, FL), and ( d ) the University of Rochester, Department of Pathology and Molecular Laboratory (Rochester, NY). Genomic DNA was isolated from 50–300 μL of buffy coat or whole blood with the QIAamp® Blood Kit (QIAGEN, Inc.), the Epicentre MasterPureTM DNA Purification Kit (Epicentre, Inc.), or the Bio-Rad InstaGeneTM Whole Blood Kit (Bio-Rad, Inc.; Table 1⇓ ). DNA was quantified by the PicoGreen® Assay (Molecular Probes) or by measuring absorbance at 260 nm. The DNA concentrations of …

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R2 = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.