Takara sybr premix ex taq kit

cDNA was reverse transcribed (30?μl), and experiments were performed following the procedure recommended by TaKaRa M-MLV reverse transcriptase (Takara Bio, Inc.) product specifications. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments were performed according to the recommended methods provided by the manufacturer of the TaKaRa SYBR® Premix Ex Taq kit (Takara Bio, Inc.). U6 and GAPDH were used as housekeeping genes for standardization. The primer sequences are listed in Supplementary Table 1. Data are presented as the fold change of downregulation or upregulation (fold value = 2^-ΔΔCt, where ΔΔCt = (Ct of the gene of interest, treated-Ct of the housekeeping gene, treated) - (Ct of the gene of interest, control-Ct of the housekeeping gene, control), and Ct was the number of threshold cycles).

Two sets of siRNA oligo nucleotides for the human PSPC1 gene corresponding to nucleotides 1257—1275 (siPSPC1) and negative control siRNA were synthesized by Shanghai GenePharma Co., Ltd and used for transfection. siRNAs were transfected into HeLa cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA), essentially as directed by the manufacturer and using a siRNA concentration of 40 nM. In short, cells were seeded into a 6-well cell culture plate, siRNA-Lipofectamine2000 complexes were added to each well after 24 h, and the medium was changed after 6 h incubation. After 18 h incubation, the attenuation of mRNA levels was detected by real-time reverse transcriptase PCR (RT-PCR). Total RNA was isolated using Trizol Reagent (Invitrogen), and 2 µg of total RNA was used for first-strand cDNA synthesis with Super Script III Reverse Transcriptase (Invitrogen). RT-PCR was performed in 20 µl using the TakaRa SYBR Premix Ex Taq Kit (TaKaRa Biotechnology, Dalian, China) and 100 ng of input cDNA template. β-actin was used as an internal standard. Primers for PSPC1 were 5′-AGACGCTTGGAAGAACTCAGA-3′ and 5′-TTGGAGGAGGACCTTGGTTAC-3′; primers for β-actin were 5′-TGCGTGACATTAAGGAGAA-3′ and 5′-AAGGAAGGC TGGAAGAGT-3′.