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Background: Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used. Results: We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements. Conclusion: The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods. [ABSTRACT FROM AUTHOR]

Background: The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters. Results: We report the results of an interlaboratory comparison of gene expression array measurements on the same microarray platform, in which the RNA amplification and labeling, hybridization and wash, and slide scanning were each individually varied. Identical input RNA was used for all experiments. While some sources of variation have measurable influence on individual microarray signals, they showed very low influence on sample-to-reference ratios based on averaged triplicate measurements in the two-color experiments. RNA labeling was the largest contributor to interlaboratory variation. Conclusion: Despite this variation, measurement of one particular breast cancer gene expression signature in three different laboratories was found to be highly robust, showing a high intralaboratory and interlaboratory reproducibility when using strictly controlled standard operating procedures. [ABSTRACT FROM AUTHOR]