RT-PCR of circularized miRNA by a pair of 5′-overlapping primers yields multimeric amplicons. (A) Alignment of 5′-overlapping primers with circular (CT) and linear (LT) forms of an miRNA. (B) Synthetic linear and circularized let-7b miRNAs (2 nM) were used as templates for reverse transcription by SSII using Primer R (80 nM), followed by PCR using the same Primer R together with Primer F. Primer R (18 nt) was complementary to nucleotides 1–18 of let-7b miRNA, while Primer F (18 nt) corresponded to nucleotides 4–21 of let-7b. The 5′-ends of Primers F and R have 15 nt of complementarity, leaving 3-nt overhangs at their 3′-ends when hybridized to each other. The PCR products were analyzed on a 3% agarose gel. Lanes 1–3 correspond to 20 cycles, and lanes 4–6 to 25 cycles of PCR. (Lanes 3,6) Negative controls, in which the RT-PCR reactions were carried out without the miRNA templates. (Lane L) A DNA ladder. (UP) Unused PCR primers.
