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Genome Biology

, 2:research0055.1

First Online: 21 November 2001Received: 27 July 2001Revised: 28 September 2001Accepted: 17 October 2001

Abstract

BackgroundAffymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization.

ResultsWe first explore the conditions for validity of the -constant mean assumption-, the key assumption underlying current normalization methods. We introduce -frequency normalization-, a -spike-in-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define -scaled frequency-, a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages.

ConclusionsScaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Electronic supplementary materialThe online version of this article doi:10.1186-gb-2001-2-12-research0055 contains supplementary material, which is available to authorized users.

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Autor: Andrew A Hill - Eugene L Brown - Maryann Z Whitley - Greg Tucker-Kellogg - Craig P Hunter - Donna K Slonim

Fuente: https://link.springer.com/



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