Determination of the transcription efficiency of horse RNA PolI promoters of different lengths. (A) Generation of virus-like luciferase reporter RNAs under the control of horse PolI promoters of various lengths. The antisense firefly luciferase coding sequence was flanked by the 5′ and 3’ noncoding sequences of the NP segment of A/equine/Maimi/1/1963; and (B) each of the constructs (100 ng) pLW1000-NPluci, pLW750-NPluci, pLW500-NPluci, pLW250-NPluci, pLW125-NPluci, and pLW0-NPluci was individually combined with pCAGGs-Miami PB1, PB2, PA, NP (100 ng of NP, 50 ng [each] of PB1, PB2, and PA), and pRL-TK (10 ng) and co-transfected into FEL cells in a 24-well plate. At 24 h after transfection, the relative luciferase activity was identified by the dual-luciferase assay. The Renilla luciferase value was used to normalize the transfection efficiencies. The relative luciferase activity associated with the pLW1000-NPluci construct was set as 100%. Each experiment was repeated three times. Statistical significance was compared with the result of the pLW1000-Npluci group, using an unpaired two-tailed Student’s t-test with GraphPad Prism 5.01 [27] (**, p < 0.01; ***, p < 0.001; NS, not significant).