Semiquantitative RT-PCR Analysis of Splicing Products

For analysis of splicing products using the pcNLmini-RI vector, semiquantitative RT-PCR analysis was carried out similarly as described previously [29 (link),32 (link)]. Briefly, HEK293T cells were transfected with vectors, and on the next day, the cells were lysed for automatic RNA extraction using a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) and QIAcube system (Qiagen). RNA samples were used for cDNA synthesis using an oligo(dT) primer. Semiquantitative PCR reactions were performed using the cDNA samples as templates and specific primer sets for all transcripts, the full and D1/A1 products, and the D1/A1-D2b/A2, D1/A1-D2/A2, and D1/A2 products [29 (link),32 (link)]. PCR amplicons were analyzed by the agarose gel electrophoresis using Metaphor agarose (Lonza Group AG, Basel, Switzerland) followed by visualization using the Amersham Imager 600 instrument (GE Healthcare, Chicago, IL, USA).