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      Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma

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          RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques.


          We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p < 10e -20) in the unamplified group have a p-value below 10e -20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e -20 < p < 10e -10) in the unamplified group have a p-value below 10e -10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics.


          We conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used.

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          Most cited references 16

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          Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: A novel neuronal marker of nerve injury.

           H Tsujino,  Y Dai,  T Ochi (2000)
          Activating transcription factor 3 (ATF3), a member of ATF/CREB family of transcription factors, is induced in a variety of stressed tissue. ATF3 regulates transcription by binding to DNA sites as a homodimer or heterodimer with Jun proteins. The purpose of this study was to examine the expression and regulation of ATF3 after axonal injury in neurons in dorsal root ganglia (DRG) and spinal cord. In naive rats, ATF3 was not expressed in the DRG and spinal cord. Following the cut of peripheral nerve, ATF3 was immediately induced in virtually all DRG neurons and motoneurons that were axotomized, and the time course of induction was dependent on the distance between the injury site and the cell body. Double labeling using immunohistochemistry revealed that the population of DRG neurons expressing ATF3 included those expressing c-jun, and in motoneurons ATF3 and c-jun were concurrently expressed after axotomy. In contrast to c-jun, ATF3 was not induced transsynaptically in spinal dorsal horn neurons. We conclude that ATF3 is specifically induced in sensory and motoneurons in the spinal cord following nerve injury and should be regarded as an unique neuronal marker of nerve injury in the nervous system. Copyright 2000 Academic Press.
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            Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth.

            The ability of neurons to regenerate an axon after injury is determined by both the surrounding environment and factors intrinsic to the damaged neuron. We have used cDNA microarrays to survey those genes induced during successful sciatic nerve regeneration. The small proline-rich repeat protein 1A (SPRR1A) is not detectable in uninjured neurons but is induced by >60-fold after peripheral axonal damage. The protein is localized to injured neurons and axons. sprr1a is one of a group of epithelial differentiation genes, including s100c and p21/waf, that are coinduced in neurons by axotomy. Overexpressed SPRR1A colocalizes with F-actin in membrane ruffles and augments axonal outgrowth on a range of substrates. In axotomized sensory neurons, reduction of SPRR1A function restricts axonal outgrowth. Neuronal SPRR1A may be a significant contributor to successful nerve regeneration.
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              Solving the riddle of the bright mismatches: labeling and effective binding in oligonucleotide arrays.

              RNA binding to high-density oligonucleotide arrays has shown tantalizing differences with solution experiments. We analyze here its sequence specificity, fitting binding affinities to sequence composition in large datasets. Our results suggest that the fluorescent labels interfere with binding, causing a catch-22. To be detected, the RNA must both glow and bind: without labels it cannot be seen even if bound, while with too many it will not bind. A simple model for the binding of labeled oligonucleotides sheds light on the interplay between binding energies and labeling probability.

                Author and article information

                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                9 October 2006
                : 7
                : 252
                [1 ]Bioinformatics Unit, Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK
                [2 ]School of Crystallography, Birkbeck College, University of London, Malet Street, London WC1 7HX, UK
                [3 ]Neural Plasticity Unit, UCL Institute of Child Health, 30 Guilford St, London WC1N 1EH, UK
                Copyright © 2006 Diboun et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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