Sybr primescript rt qpcr kit

Total RNA was extracted from cultured cells or tissues using TRIzol Reagent (Invitrogen, USA) and transcribed into cDNA using a PrimeScriptTM RT Reagent Kit (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s instructions. qRT-qPCR was performed with a SYBR PrimeScript RT-qPCR Kit (Takara, USA) for 40 cycles at 95°C for 5 s and 60°C for 30 s on a Light Cycler 480 Real-Time PCR System (Roche, Switzerland). All primer sequences used in this study are listed in the supplemental information (Table S2).

TRIzol^® reagent (Invitrogen, Thermo Fisher Scientific, USA) was used to extract total RNA from cultured cells, and tissue samples as protocol showed. PrimeScript RT Reagent kit synthesized complementary DNA (Takara Bio, Inc., Otsu, Japan). RT-PCR was performed using the SYBR Prime Script™ RT-qPCR kit (Takara Bio, Inc., Otsu, Japan) on an ABI 7500 system (Applied Biosystems, Thermo Fisher Scientific, USA). The 2^−ΔΔCT method was used for calculation, and each sample was done in triplicate. The primer sequences were listed below: CARLo-7, forward: 5'-GCTGC AGAAG GTCCG AAGAA-3', reverse: 5'-TTCAC CACGT CCAGT TGCTT-3'. GAPDH, forward: 5'-GGAAA GCTGT GGCGT GAT-3', reverse: 5'-AAGGT GGAAG AATGG GAGTT-3'.

Total RNA was isolated from placenta samples and HTR-8/SVneo cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.). Subsequently, RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using a PrimeScript RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocol. The thermocycling conditions were as follows: 95°C for 10 min, 42°C for 2 min, 37°C for 15 min, 85°C for 5 sec and 4°C for 30 min. qPCR was performed using a SYBR PrimeScript RT-qPCR kit (Takara Bio, Inc.) on a Roche Light-cycler 480 real-time qPCR System (Roche Applied Science), according to the manufacturer's protocol. The thermocycling conditions of the qPCR were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Primers for miR-320a, U6, IL-4 and GAPDH are presented in Table II. The primers were designed using Primer Premier software (version 5.0; Premier Biosoft International, Inc.), and purchased from Takara Biotechnology Co., Ltd. Expression level of miR-320a was normalized to the U6 expression level. IL-4 expression was normalized to GAPDH expression. Relative expression was calculated using the 2^−∆∆Cq method (21 (link)).