FIGURE 3.

Detection of mature miR-675 in newborn mice. This RPA analyzed 50 μg of total RNA derived from murine 3T3 cells (lane 2), a newborn (NB) mouse (lane 3), mock-transfected 293T cells (lane 4), or 293T cells transfected with a mouse H19 (mH19) expression plasmid (lane 5). The 31-nt-long single-stranded RPA probe used consisted of a 23-nt stretch complementary to the predicted mature mouse miR-675, a 5-nt 5′ extension complementary to part of the predicted terminal loop of the pre-miR-675 RNA hairpin (Fig. 1A), and finally a 3-nt nonspecific 5′ tag consisting of “GGG.” The probe was prepared by in vitro transcription, using T7 RNA polymerase, in the presence of α-³²P-CTP. This RPA was performed using a miRVana miRNA detection kit (Ambion), as described by the manufacturer. (M) DNA markers.