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Abstract Background Various methods for estimating protein expression levels are known. The level of correlation between these methods is only fair, and systematic biases in each of the methods cannot be ruled out. We here investigate systematic biases in the estimation of gene expression rates from microarray data and from abundance within the Expressed Sequence Tag (EST) database. We suggest that length is a significant factor in biases to measured gene expression rates. As a specific example of the importance of the bias of expression rate with length, we address the following evolutionary question: Does the average <it>C. elegans </it>protein length increase or decrease with expression level? Two different answers to this question have been reported in the literature, one method using expression levels estimated by abundance within the EST database and another using microarrays. We have investigated this issue by constructing the full protein length versus expression curve for <it>C. elegans</it>, using both methods for estimating expression levels. Results The microarray data show a monotonic decrease of length with expression level, whereas the abundance within the EST database data show a non-monotonic behavior. Furthermore, the ratio of the expression level estimated by the EST database to that measured by microarrays is not constant, but rather systematically biased with gene length. Conclusions It is suggested that the length bias may lie primarily in the abundance within the EST database method, being not ameliorated by internal standards as it is in the microarray data, and that this bias should be removed before data interpretation. When this is done, both the microarray and the abundance within the EST database give a monotonic decrease of spliced length with expression level, and the correlation between the EST and microarray data becomes larger. We suggest that standard RNA controls be used to normalize for length bias in any method that measures expression.</p

Microarray and EST database estimates of mRNA expression levels differ: The protein length versus expression curve for C. elegans