Cytokine Protocols pp 55-70
Quantitative Analysis of miRNA Expression in Epithelial Cells and Tissues
Reliable detection of the microRNA (miRNA) precursor and mature form expression levels is a fundamental starting block for more focused studies of the biogenesis and functional roles of these important post-transcriptional modulators of gene expression. Building on our expertise with miRNA expression programs downstream of TGF-β/Smad signaling in homeostasis as well as in pathological conditions associated with epithelial tissues, we present a series of detailed and broadly applicable protocols for expression profiling of the mature miRNA forms using quantitative real-time PCR TaqMan, both single assays or low-density arrays. We next highlight key steps necessary for the detection of primary precursors of miRNAs (pri-miRNAs) to address the initial steps of miRNA biogenesis, and we finally review some most widely used computational algorithms for miRNA target prediction used to complement experimental identification of the target mRNAs and proteins.
Key wordsmicroRNA miRNA miRNA expression Pri-miRNA expression Quantitative real-time PCR TaqMan Array MicroRNA Card TaqMan MicroRNA Assay Primer3 SYBR Green PCR miRNA target prediction TGF-β Epithelial cell Epithelial tissue
Laser capture microdissection
Primary precursor of miRNA
Quantitative real-time polymerase chain reaction
TaqMan low-density array(s)