Research article

Evaluation of a new high-dimensional miRNA profiling platform

Julie M Cunningham^1* , Ann L Oberg^2* , Pedro M Borralho³ , Betsy T Kren⁴ , Amy J French¹ , Liang Wang¹ , Brian M Bot² , Bruce W Morlan² , Kevin AT Silverstein⁵ , Rod Staggs⁵ , Yan Zeng⁶ , Anne-Francoise Lamblin⁵ , Christopher A Hilker¹ , Jian-Bing Fan⁷ , Clifford J Steer⁴ and Stephen N Thibodeau¹

¹  Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, Minnesota, USA

²  Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota, USA

³  iMed.UL, Faculty of Pharmacy, University of Lisbon, Lisbon 1649-003, Portugal

⁴  Departments of Medicine and Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, USA

⁵  Biostatistics and Informatics, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

⁶  Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA

⁷  Illumina Inc. 9885 Towne Centre Drive, San Diego, California, 92121, USA

author email corresponding author email* Contributed equally

BMC Medical Genomics 2009, 2:57doi:10.1186/1755-8794-2-57

Published:	27 August 2009

Abstract

Background

MicroRNAs (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.

Methods

We 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.

Results

Reproducibility 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.

Conclusion

These 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.