An Improved Workflow for miRNA Expression Profiling Using Ion Semiconductor Sequencing

MicroRNAs (miRNAs) are non coding RNA molecules with a size range of 18 to 24 nucleotides that have important functions in regulating numerous cellular processes. Several technologies such as microarrays, real-time PCR and second-generation deep sequencing have been used to profile miRNA expression and characterize variances in miRNA species. This study focuses on miRNA expression profiling using ion semiconductor sequencing with an improved workflow that utilizes multi-channel microfluidics to isolate miRNAs from total RNA. This microfluidics approach replaces polyacrylamide gel electrophoresis for size fractionation and enrichment of miRNAs commonly used in sequencing protocols. We compared miRNA expression profiles of commercially available preparations of lung and brain total RNA obtained by ion semiconductor sequencing, second-generation sequencing and real-time PCR array. We show that ion semiconductor sequencing using our improved workflow with the microfluidics device produced similar miRNA expression profiles compared to polyacrylamide gel based methods. We also detected similar numbers of miRNAs compared to second-generation deep sequencing. Finally, we were able to validate our results using a commercially available real-time PCR based miRNA array. Our workflow streamlines and significantly improves the utilization of semiconductor sequencing for miRNA profiling both in a research and clinical environment.