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. 2017 Feb;23(2):161–168. doi: 10.1261/rna.058024.116

FIGURE 2.

FIGURE 2.

Primer extension assay to determine the 5′-terminal position of 5′-SHOT-RNAHisGUG. (A) The cloverleaf secondary structure of 5′-SHOT-RNAHisGUG used as a primer extension template. The 5′-end-labeled 20-nt primer, which was hybridized to the D-arm of tRNA, is shown as a black solid line; nascent cDNA synthesized from the primer is indicated as a gray dotted line. Reverse transcription from the primer terminates at np 1 or −1 to yield a cDNA band with a length of 25 or 26 nt, respectively. (B) Synthetic mature tRNAHisGUG containing either G1 or G−1, or a 30- to 50-nt small RNA fraction of BT-474 cells were subjected to a primer extension assay for an analysis of the 5′-terminal position of 5′-SHOT-RNAHisGUG. An assay without template RNA was also performed as a negative control experiment.