Characterization of the processing of the shRNA-intron in Cos and 293T cells. (A) The predicted processing of the shRNA-intron in vivo as well as the primers and a stem-loop primer are schematically illustrated. Primer 1 detected the unspliced mRNA and a spliced out intron, and primer 2 detected the siRNA. (B) The presence of siRNA and the intron was examined by end-point RT-PCR 48 h after the transfection of Cos cells (C) and 293T cells (T) with vectors expressing shRNA1 or shRNA1-intron. The empty pRNA-CMV3.1-Neo vector was used as a control. (C) The siRNA levels detected in cells transfected with the shRNA2 and shRNA2-intron vectors. (D) The levels of siRNA compared to those of the intron produced from the shRNA1-intron or shRNA2-intron vector in Cos cells and 293T cells were analyzed by RT-qPCR (each normalized to G3PDH mRNA). The normalized values of the siRNA/intron levels were set to 100 in Cos cells.