Tempase hot start 2 master mix

Cell lysis was performed by adding TRIzol Reagent (Ambion, Carlsbad, CA). After homogenization of the cell lysates, total RNA was extracted by phase separation with chloroform and precipitation in isopropanol. cDNA was synthesized from 0.5 μg RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) in 10-μL reactions. To investigate splicing patterns of the minigene PCCA pseudoexon, the region from PCCA exon 14 to the bovine growth hormone (BGH) termination sequence of the pcDNA3.1(+) backbone was amplified (PCCA.ex14.F: 5′-ACCCCTACAAGTCTTTTGGTTTAC-3′ and BGH.R: 5′- AACTAGAAGGCACAGTCGAGGCTG-3′) from 1 μL cDNA using TEMPase Hot Start 2× Master Mix (Ampliqon, Odense M, Denmark) in 10-μL PCR reactions. To investigate the splicing patterns of the endogenous PCCA pseudoexon, the region from PCCA exon 14 to exon 15 was amplified (PCCA.ex14.F and PCCA.ex15.R: 5′-TTCCTGGTTGGATGCCACTG-3′). (PCR program: 95°C for 15 min, 30 cycles at 95°C for 30 s, 60°C (minigene)/51°C (endogenous) for 30 s, and 72°C for 20 s, followed by 72°C for 5 min.) PCR samples were visualized on 1.5% SeaKem LE agarose (Lonza, Rockland, ME) gels containing 0.5× GelRed (Biotium, Fremont, CA), and pseudoexon inclusion was quantified from molar ratios between PCR products using the Fragment Analyzer system (Advanced Analytical Technologies, Ames, IA).