THESIS
2021
1 online resource (xi, 83 pages) : illustrations (chiefly color)
Abstract
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...[
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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.
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