Figure 3

Small RNAs derived from the non-coding hY4 RNA present in plasma. (A) The predicted secondary structure of hY4 is shown in dot-bracket notation above the sequence and the reads mapping to the 5p and 3p arms indicated below (numbers refer to the reads detected in sample 1a). The positions of the most abundant 5p and 3p reads (and much less frequent short reads) are indicated by arrows adjacent to the hY4 structure. (B) Custom Taqman small RNA assays targeting the hY4-5p or 3p RNAs corresponding to the most abundant reads amplified products several threshold cycles before individual microRNAs (eg miR-22 in sample 1a). (C) RT-PCR with primers specific for the putative hY4 fragments and performed upon RNA that had been polyadenylated, amplified products with lengths consistent with the presence of the small RNA templates detected in the sequencing rather than full length hY4 RNA. A product of the predicted size (79 bp) was detected with the hY4 5p primer, whereas a longer product of 143 bp would have been amplified from full length hY4 RNA. M: Marker, sizes in bp; Lane 1, hY4 5p; Lane 2, No RT; Lane 3, hY4 3p; Lane 4, No RT.