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Evaluation of microarray preprocessing algorithms based on concordance with RT-PCR in clinical samples.

ABSTRACT: Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been performed.We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental data sets: one comprising 84 genes in 36 colon biopsies, and the other comprising 75 genes in 29 cancer cell lines. We evaluated consistency using the Pearson correlation between measurements obtained on the two platforms. Also, we introduce the log-ratio discrepancy as a more relevant measure of discordance between gene expression platforms. Of nine preprocessing algorithms tested, PLIER+16 produced expression values that were most consistent with RT-PCR measurements, although the difference in performance between most of the algorithms was not statistically significant.Our results support the choice of PLIER+16 for the preprocessing of clinical Affymetrix microarray data. However, other algorithms performed similarly and are probably also good choices.

SUBMITTER: Gyorffy B

PROVIDER: S-EPMC2680989 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Evaluation of microarray preprocessing algorithms based on concordance with RT-PCR in clinical samples.

Gyorffy Balazs B Molnar Bela B Lage Hermann H Szallasi Zoltan Z Eklund Aron C AC

PloS one 20090521 5

<h4>Background</h4>Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been performed.<h4>Methodology/principal findings</h4>We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental da ...[more]

PMID: 19461970

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Project description:BackgroundDengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.MethodsThe DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.ResultsThe RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.ConclusionsOur results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.

Project description:Background:Kyasanur Forest Disease (KFD), a tick borne flavivirus, which was earlier endemic to Karnataka state, India, has been confirmed and detected from neighboring states of Tamil Nadu, Maharashtra, Goa and Kerala states in India. Increased human and vector surveillance therefore becomes essential for the identification of KFD affected regions and control of further spread of the disease. Currently, available KFD detection assays include realtime RT-PCR and nested RT-PCR assays. Here we describe the development of a sensitive single step RT-PCR assay for the detection of KFD viral RNA. This can be easily used in any BSL-2 laboratory for screening of KFD suspected cases or for differential diagnosis of viral hemorrhagic fever panel. Method:Three primer sets were designed and checked for sensitivity using known dilutions of KFD viral RNA (Ranging from 106 copies to 10 copies). The primer set (2) was found to be most sensitive was selected and tested for specificity for Kyasanur forest disease virus (KFDV) by testing against zika, dengue, chikungunya, crimean congo hemorrhagic fever (CCHF), yellow fever, japanese encephalitis (JE) and west nile viruses. A total of 104 samples (human, monkey and tick positive and negative samples) were tested using this assay. Result:No false positive or false negative results were seen for human, monkey or tick samples. The assay was specific for KFD and could detect upto 100 copies of KFD viral RNA. Discussion and conclusion:The previously published sensitive real time RT-PCR assay requires higher cost in terms of reagents and machine setup and technical expertise has been the primary reason for development of this assay. A single step RT-PCR is relatively easy to perform and more cost effective than real time RT-PCR in smaller setups in the absence of Biosafety Level-3 facility. This study reports the development and optimization of single step RT-PCR assay which is more sensitive and less time-consuming than nested RT-PCR and cost effective for rapid diagnosis of KFD viral RNA.

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Evaluation of microarray preprocessing algorithms based on concordance with RT-PCR in clinical samples.

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Evaluation of microarray preprocessing algorithms based on concordance with RT-PCR in clinical samples.

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