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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
Published on: August 3, 2011
Evaluation of a new high-dimensional miRNA profiling platform.
Julie M Cunningham1, Ann L Oberg, Pedro M Borralho
1Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, Minnesota, USA. cunningham.julie@mayo.edu
BMC Medical Genomics
|August 29, 2009
Summary
This study demonstrates that the Illumina miRNA profiling platform accurately quantifies microRNA (miRNA) expression using minimal RNA input. The assay shows excellent reproducibility and sensitivity for miRNA analysis.
Area of Science:
- Molecular Biology
- Genomics
Background:
- MicroRNAs (miRNAs) are crucial regulatory molecules in eukaryotes.
- Accurate quantification of miRNA expression is vital for functional studies.
Purpose of the Study:
- To evaluate a novel miRNA profiling platform based on Illumina's DASL chemistry.
- To assess the reproducibility and sensitivity of the platform using varying RNA input.
Main Methods:
- Utilized Illumina's DASL chemistry for miRNA profiling.
- Tested the platform with total RNA from colon cancer patients and cell lines.
- Assessed reproducibility across replicates and varying RNA input amounts (25-800 ng).
Main Results:
- High reproducibility observed between sample replicates (Spearman's correlation 0.91-0.98) and across different days (0.84-0.98).
- No significant effect on data quality was found across a wide range of RNA input (25-800 ng).
- Nonlinear biases were detected, necessitating abundance-dependent normalization.
Conclusions:
- The Illumina miRNA platform enables sensitive miRNA profiling with minimal starting material.
- The system demonstrates excellent performance characteristics for miRNA analysis.
- Abundance-dependent normalization is recommended for optimal results.
