Blog
About

  • Record: found
  • Abstract: found
  • Article: not found

Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Read this article at

ScienceOpenPublisherPMC
Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, ρ-value, fold-change, cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρ>0.80 consistently observed when quality scores are applied.

      Related collections

      Most cited references 28

      • Record: found
      • Abstract: not found
      • Article: not found

      Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.

      There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

         S Bustin (2000)
        The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
          Bookmark
          • Record: found
          • Abstract: not found
          • Article: not found

          Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

           S Bustin (2002)
          The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.
            Bookmark

            Author and article information

            Affiliations
            [1 ]NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research. 219 Fort Johnson Rd., Charleston, SC, 29412. USA.
            Author notes
            Jeanine S. Morey, NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research. 219 Fort Johnson Rd., Charleston, SC, 29412. USA. Jeanine.Morey@ 123456noaa.gov
            Journal
            Biol Proced Online
            Biological Procedures Online
            Biological Procedures Online
            1480-9222
            2006
            12 December 2006
            : 8
            : 175-193
            1779618
            17242735
            m126
            10.1251/bpo126
            Copyright © December 12, 2006, JS Morey et al. This paper is Open Access and is published in Biological Procedures Online under license from the authors. Copying, printing, redistribution and storage permitted.
            Categories
            Research Article

            Comments

            Comment on this article