Miscript sybr green real time pcr kit

Total RNA was extracted from mouse endometrial tissues or cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. RT of cDNA was conducted using the miScript II Reverse Transcription kit with miScript HiSpec Buffer (Qiagen, Inc., Valencia, CA, USA,). Briefly, RNA was mixed with miScript HiSpec Buffer, RNAse-free water and miscript Reverse Transcriptase Mix, and was incubated at 37°C for 60 min and 95°C for 5 min. qPCR was conducted with the miScript SYBR-Green Real Time PCR kit (Qiagen, Inc.) for miRNA detection and SYBR Premix Ex Taq. The specific primers for mmu-miR-96, U6, Bcl2, decidual/trophoblast prolactin-related protein (dtPRP) and β-actin were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table I). The qPCR master mix (15 µl) contained 7.5 µl SYBR Premix Ex Taq, 0.6 µl primers, 1.2 µl cDNA and 5.1 µl diethylpyrocarbonate-treated H₂O. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec; 40 cycles at 95°C for 5 sec (denaturation) and 60°C for 30 sec, followed by 72°C for 5 sec. Experiments were performed in triplicate. Data obtained from qPCR were analyzed using the 2-^ΔΔCq method (26 (link)).

Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instruction. RNA was reverse transcribed using the miScript II Reverse Transcription Kit with miScript HiSpec Buffer (Qiagen). All PCR reactions were carried out in an ABI 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the miScript SYBR Green Real-Time PCR Kit (Qiagen). After completion of PCR amplification, relative expression of the tested genes was calculated based on the 2-ΔΔCt method. The mRNA level of GAPDH was measured as an internal control. Each sample was run independently in triplicate. The RT-PCR primers used are listed in Table S1.