Sensifast sybr buffer

Total RNA from the heart of the chick embryo was isolated using the E.Z.N.A. Total RNA Kit 1 (R6834-02, Omega Bio-Tek). This RNA was treated with DNase I (E1091-02, Omega Bio-Tek) prior to cDNA synthesis. cDNA was created using the qScript cDNA synthesis kit (Cat# 95047-100, Quanta Biosciences). cDNA was quantitated using the Quanti-iT Oligreen ssDNA assay kit (011492, Invitrogen) and a fluorescent plate reader relative to a set of ssDNA standards. 10 ng of cDNA was added to each PCR tube for measurement. Quantitative PCR was accomplished using a Rotorgene Instrument (Qiagen) using a SensiFAST SYBR buffer (Bio-98002, Bioline). Cycling parameters used a 60°C annealing temperature and 72°C temperature for elongation and data collection. Primers were designed for chick marker genes using NCBI Primer-Blast and selected for pairs that gave melting curves with a single peak. Effective primer sequences are shown in Table 2.

Total RNA from mice embryo hearts was treated with DNaseI (E1091–02, Omega-BioTek). cDNA was transcribed using the iScript cDNA synthesis kit (Bio-Rad). Total cDNA was used to normalize quantitative PCR reactions as we have previously published (Mercado-Pimentel; Tavares). cDNA was sensitively measured in each reaction using Quanti-iT Oligreen ssDNA reagent (011492, Invitrogen) with a fluorometer (Turner Biosystems). Equal aliquots of cDNA (10ng) were dispensed in each PCR tube for measurement. Quatitative PCR was performed on a Rotorgene Instrument (Qiagen) using a SensiFAST SYBR buffer (Bio-98002, Bioline). Cycling parameters used a 60°C annealing temperature and 72° C for elongation and data collection. Primers were designed for mouse and human marker genes using NCBI Primer-Blast and selected for pairs that gave melting curves with a single peak. Primers sequences are displayed in Table 1.