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BMC Medical Genomics

, 2:57

First Online: 27 August 2009Received: 12 January 2009Accepted: 27 August 2009


BackgroundMicroRNAs miRNAs are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.

MethodsWe evaluated a new miRNA profiling platform that utilizes Illumina-s existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina-s miRNA profiling application.

ResultsReproducibility between sample replicates within a plate was good Spearman-s correlation 0.91 to 0.98 as was the plate-to-plate reproducibility replicates run on different days Spearman-s correlation 0.84 to 0.98. To determine whether quality data could be obtained from a broad range of input RNA, data obtained from amounts ranging from 25 ng to 800 ng were compared to those obtained at 200 ng. No effect across the range of RNA input was observed.

ConclusionThese results indicate that very small amounts of starting material are sufficient to allow sensitive miRNA profiling using the Illumina miRNA high-dimensional platform. Nonlinear biases were observed between replicates, indicating the need for abundance-dependent normalization. Overall, the performance characteristics of the Illumina miRNA profiling system were excellent.

Electronic supplementary materialThe online version of this article doi:10.1186-1755-8794-2-57 contains supplementary material, which is available to authorized users.

Julie M Cunningham, Ann L Oberg contributed equally to this work.

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

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