cytoplasm, these precursor microRNAs are cleaved by the essential
enzyme Dicer, into single-stranded functional units [4].
MicroRNAs were originally identified fromC. elegansgenetic
screens for defects in developmental transitions [5, 6]. Within the
last 7–10 years, there has been an explosion of studies documenting
the expanded roles of miRNAs, which now include organogenesis,
stem cell maintenance and brain and cardiac morphogenesis and
tissue regeneration [7–12]. MicroRNAs regulate gene expression at
the posttranscriptional level, by binding to the 3^0 UTR of target
mRNAs and inhibiting protein translation. An individual micro-
RNA is capable of regulating a large set of target genes, ranging
from 10 to 500 target mRNAs [13, 14]. Thus, to understand
microRNA biology, one must ideally detect both microRNA and
target gene.
Detection of microRNAs is confounded by both its small size
and low expression levels. The use of locked nucleic acid probes
targeting the mature sequence has been successfully used to detect
highly abundant microRNAs [15]. However, often these protocols
are incompatible with simultaneous detection of a potential target
gene. Advanced Cell Diagnostics (ACD) has recently pioneered
probe design and amplification strategies that maximize signal
intensity while suppressing background noise (Fig.1, https://
acdbio.com/). Here, I describe this approach on adult zebrafish
heart cryosections detecting microRNA-101a andfosabmRNA.Fig. 1Experimental paradigm of a multiplex RNAscope amplification of ISH
signal. Sections are hybridized with multiple probes designed for two different
target genes. Through a series of amplification chemistry with propriety technol-
ogy, each probe signal is amplified. Addition of chromogenic substrates enables
detection of RNAs198 Viravuth P. Yin