Easyscript first strand cdna synthesis kit

Total RNA from cells and tissues were isolated using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA (1 µg) was reverse-transcribed into cDNA using the EasyScript First-strand cDNA Synthesis kit (Beijing Transgen Biotech Co., Ltd., Beijing, China). The analysis of miR-195-5p levels was performed by qPCR analysis according the protocol of the SYBR-Green II kit (Takara Biotechnology Co., Ltd., Dalian, China). cDNA (1 µl), 10 µl SYBR Green mixture, 0.8 µl primers (10 µM) and 8.2 µl ddH₂O were mixed. The procedures of PCR were described as follow: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 20 sec. The relative level of miR-195-5p was normalized by U6 small nucleolar RNA, which was used as the housekeeping gene. The gene expression level was calculated using the 2^−ΔΔCq method (19 (link)). All analyses were performed in triplicate. The primers were designed using Primer Premier 5 (Premier Biosoft International, Palo Alto, CA, USA. The sequences of the RT primers were as follows: miR-195-5p, 5′-TTCCGATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT-3′; U6, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAATATG-3′. The nucleotide primers used for qPCR were as follows: miR-195-5p, forward, 5′-GCGATAGCAGCACAGAAATA-3′; U6, forward 5′-GCGCGTCGTGAAGCGTTC-3′ and universal reverse, 5′-GTGCAGGGTCCGAGGT-3′.

The T47D and MCF-7 cell lines at a density of 5× 10⁴cells/ml were cultured into six-well plates followed by incubation at 37°C in CO₂ incubator. After 24 hr of incubation, the cells were treated with ZnONPs for another 24 hr. Total RNA isolation from the T47D and MCF-7 cell lines was conducted using the EasyPure® RNA Kit (TransGen Biotech ER101-01, China) according to the manufacturer’s protocol. The cDNA synthesis was done using the EasyScript® First-Strand cDNA Synthesis Kit (TransGen Biotech AE301-02, China) based on the manufacturer’s protocol. The sequence of forward and reverse specific primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), KAI-1, and NM23 genes was designed using Primer Express Software version 3.0 (Table 1) and verified using BLAST analysis at NCBI (www.ncbi.nlm.nih.gov/blast). The GAPDH was used as the housekeeping gene for normalization within a given sample. The quantitative real-time polymerase chain reaction was performed in ABI7300 real-time PCR system (Applied Biosystems Company, USA) with SYBR GREEN^® (Non-specific DNA-binding factors) based on the manufacturer’s protocol. The relative quantifications of NM23 and KAI-1 mRNA expression levels were compared with the housekeeping gene (GAPDH) using the 2^−ΔΔCt method 16 .

Total RNAs were isolated by one-step method using TRIzol reagent according to the instruction. The RNA samples were pretreated with RNase-free DNase, and 1 µg RNA of each sample was reverse-transcribed to synthesize first-strand cDNA using EasyScript First-Strand cDNA Synthesis Kit (TransGen Biotech, China). Then the cDNA templates were amplified by real-time quantitative PCR using Maxima SYBR Green/Fluorescein QPCR Master Mix (MBI, Canada) on an ABI-7300 PCR System (Applied Biosystems, USA). PCR reaction started with 1 cycle of 95°C for 10 min, followed by 40 cycles of three steps as 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The fluorescence data were collected at 72°C step. The mRNA expression of the target gene was normalized to the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The mRNA relative expression levels of target gene were represented as 2^-ΔΔCt, which were obtained by the software SDS v3.2 of ABI-7300 PCR System. PCR primers were designed using Primer Express Software V3.0 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Sequences of primers were as follows: