A High-Throughput MicroRNA Expression Profiling System
As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings.
Key wordsNoncoding RNAs miRNAs High-throughput miRNA labeling miRNA capture
This work was supported in part by NIH grants R01CA149109 and R01GM099811 and Connecticut Stem Cell Research Fund (09SCBYALE27). We thank Eric Miska, Ezequiel Alvarez-Saavedra, Justin Lamb, David Peck, Hao Zhang, and Judy Wang for help during the process of establishing this protocol.