Micro RNAs (miRNA), an important class of short non-coding RNAs, are involved in most of the key cellular functions via regulating mRNA expression. The study of miRNAs not only can deepen our understandings of cellular gene regulation, but also unveil novel therapeutic targets and treatments. Advanced sequencing and qPCR methods have been developed for the quantification of miRNAs. Due to extensive posttranslational modification of miRNAs and sequence-activity bias of miRNAs, the amounts of miRNAs cannot be directly applied to understand the mRNA regulatory activities of the miRNAs. Therefore, miRNA activity detection relies on the direct measurement of target mRNA expression change. However, current miRNA activity assays only report the target mRNA expression difference with or without...[ Read more ]

Micro RNAs (miRNA), an important class of short non-coding RNAs, are involved in most of the key cellular functions via regulating mRNA expression. The study of miRNAs not only can deepen our understandings of cellular gene regulation, but also unveil novel therapeutic targets and treatments. Advanced sequencing and qPCR methods have been developed for the quantification of miRNAs. Due to extensive posttranslational modification of miRNAs and sequence-activity bias of miRNAs, the amounts of miRNAs cannot be directly applied to understand the mRNA regulatory activities of the miRNAs. Therefore, miRNA activity detection relies on the direct measurement of target mRNA expression change. However, current miRNA activity assays only report the target mRNA expression difference with or without the presence of a miRNA. The lack of referencing negative miRNA and positive miRNA for data normalization renders such assays unsuitable for accurate miRNA activity measurement, let alone miRNA activity comparison across cell lines. With the growing demand for miRNA activity profiling for therapeutic purposes, there is a need of establishing both positive and negative referencing miRNAs for miRNA activity assays.

Previous results collectively concluded that all miRNAs have a strong cell-type preference, thus it is unlikely a single miRNA can be used as referencing miRNA. Based on the miRNA sequencing database and the reports on artificial miRNAs, we hypothesized that referencing miRNAs can be built in the form of cocktails by a set of miRNAs with low cell-to-cell activity fluctuations. In this investigation, we first extracted candidate miRNAs from various databases and performed activity screening on various types of cell lines. We successfully built a negative miRNA cocktail using artificial miRNAs that have no activities in human cells and have confirmed that this negative cocktail can serve as a robust reference for miRNA activity measurement. We have also screened out four candidates of positive miRNA cocktails. With two levels of signal normalization, we have explored the prototype of an advanced miRNA activity assay for reporting miRNA activities across cell lines. In our future experiment, we will continue to validate these cocktails for miRNA activity measurement on primary cell lines. Because of the important role of miRNA in cell biology and broad potential in the biomedical field, we believe these referencing miRNA cocktails and the advanced assay will allow us to build miRNA activity profiles of normal and diseased cells, broadening our understanding of miRNA function and providing new perspectives for diagnostics and therapeutics.