Hotstar hifidelity taq polymerase kit

Human lung tissue (8 donors) without overt disease, but not suitable for transplant, was obtained from the Life Alliance Organ Recovery Agency according to institutional review board guidelines regarding consent and de-identification of individual donors (demographic characteristics, see Table 1). Peripheral lung tissue was excised, chopped into ~ 5 mm fragments, and snap frozen in liquid N₂. For preparation of total RNA, tissue was ground in a mortar and pestle under liquid N₂ and the powder immediately extracted using E.Z.N.A. HP Total RNA Isolation Kit (Omega Bio-Tek, Norcross, GA). Total RNA was treated with DNase I and reverse transcribed using AMV First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA) with a primer specific in exon 50 of the CACNA1C transcript 3′ UTR (see Table 1 for primers sequences). cDNA was amplified by 35 cycles of PCR using Hotstar Hifidelity Taq polymerase kit (Qiagen, Valencia, CA) with primers specific for exons surrounding known spice sites (Table 2) and the PCR reactions cloned into pGEMTeasy. cDNA in random individual colonies was isolated and sequenced.