A Highly Sensitive Target-Primed Rolling Circle Amplification
(TPRCA) Method for Fluorescent in Situ Hybridization
Detection of MicroRNA in Tumor Cells
The
ability to detect spatial and temporal microRNA (miRNA) distribution
at the single-cell level is essential for understanding the biological
roles of miRNAs and miRNA-associated gene regulatory networks. We
report for the first time the development of a target-primed RCA (TPRCA)
strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level.
This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free
3′ terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands
of complementary segments. After hybridization with fluorescent detection
probes, target miRNA molecules can be visualized with ultrahigh sensitivity.
Because the RCA reaction can only be initiated by the free 3′
end of target miRNA, the developed strategy offers the advantage over
existing ISH methods in eliminating the interference from precursor
miRNA or mRNA. This strategy is demonstrated to show high sensitivity
and selectivity for the detection of miR-222 expression levels in
human hepatoma SMMC-7721 cells and hepatocyte L02 cells. Moreover,
the developed TPRCA-based ISH strategy is successfully applied to
multiplexed detection using two-color fluorescent probes for two miRNAs
that are differentially expressed in the two cell lines. The results
reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and
clinical diagnostics.