Quantitative real time rt pcr analysis kit

The total RNA was isolated from 50 blastocysts using TRIzol reagent, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using a Quantitative Real-Time RT-PCR Analysis Kit (Bio-Rad, Munich, Germany). The complementary DNA was synthesized using 1 µg of RNA, oligo (dT) primers, and the AMV First Strand cDNA Synthesis Kit (ROCHE). The quantitative PCR amplification was performed using SYBR^® Green EX Taq™ (TaKaRa) and an RG-6000 Real-Time PCR detection system (Corbett Research Co., Mortlake, Australia). The samples were run in triplicate. The relative gene expression was calculated using the comparative threshold cycle (Ct) method, with GAPDH as the reference gene. The thermocycling conditions were as follows: 95 °C for 10 min; followed by 35 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. Fluorescence was measured once. Primer sets for each gene are listed in Table 1.

gDNA was amplified in a 50 μL PCR reaction containing 5.0 units TaKaRa Ex Taq polymerase (Takara, Shiga, Japan), PCR amplification was carried out as follows: 1 cycle of denaturation at 94°C for 5 min; 40 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 30 sec; and a final extension at 72°C for 5 min. Aliquots (10 μL) of PCR products were fractionated on 0.8% or 1.2% agarose gels and stained with RedSafe™ Nucleic Acid Staining Solution (Intron, Seongnam, Korea).
Total RNA was isolated from brain, spleen, kidney, lung, heart, liver, and muscle using the TRIzol reagent, and real-time RT-PCR reactions were performed using the Quantitative Real-Time RT-PCR Analysis kit (Bio-Rad, Munich, Germany). RT-PCR involved an initial denaturation (95°Cfor 10 min), followed by 35 cycles of amplification and quantification (95°C for 10 sec, 55°Cfor 30 sec, and 72°C for 30 sec, with a single fluorescence measurement). The experiment was repeated three independent times. The primer sets for each gene are listed in Table 5.