The miR-ID approach. (A) Circularization of a miRNA carrying 5′-phosphate (5′-p) and 3′-hydroxyl (3′-OH) ends by enzymatic ligation (Step 1). (B) Reverse transcription of the circularized miRNA (RTC) by extension of an RT primer, producing a cDNA consisting of tandem repeats of the sequence complementary to the miRNA (Step 2). Examples are shown of RT primers having various alignments relative to the ligation site. (C) qPCR amplification of segments of the multimeric cDNA products of RTC using 5′-overlapping primers together with a nonspecific dye such as SYBR Green (Step 3). (D) Examples of possible alignments of the 5′-overlapping PCR primer pairs with monomer units of the multimeric cDNA, which include straddling the boundaries between these units.
