Icycler

The mRNA levels of target genes were determined using SYBR Green supermix (Bio-Rad) on a Bio-Rad iCycler and analyzed by an accompanying software (Bio-Rad Laboratories). All reactions were run in triplicates using approximately 50 ng/ml of cDNA obtained as described above.
The primer sets used were as follows: 5′-CTAGG AGATTTGTTTGGCGTGCC-3′ and 5′GCCGTTTTCG ACCCTGAGAG-3′ for p21; and 5′-TCTCCTCTGACTT CAACAGC-3′ and 5′GAAATGAGCTTGACAAAGTG-3′ for GAPDH. Taqman real-time QRT-PCR was performed on the Bio-Rad iCycler MyiQ for quantitation of hTERT mRNA using primers and probes (sense primer, 5′TGACACCTCACCTCACCCAC-3′, anti-sense primer, 5-CACTGTCTTCCGCAAGTTCAC-3′, and Taqman probe, 5′ACCCTGGTCCGAGGTGTCCCTGAG-3′) as previously reported [39 (link), 49 (link), 50 (link)]. The quantitative data were normalized by GAPDH and quantification of relative expression levels were estimated using the ΔΔCT method. Results were presented as relative fold change. Water was used as a negative control.

Total RNA was isolated using the RNeasy Mini-kit (Qiagen, Valencia, CA USA). Single-stranded cDNA was subsequently synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA USA). Expression of GPX7 was evaluated for 45 gastric carcinomas and their matched normal gastric mucosae adjacent to cancers. The GPX7 primers (forward 5′-AACTGGTGTCGCTGGAGAAG-3′ and reverse 5′-AAACTGGTTGCAGGGGAAG-3′) were designed using the online software, Primer 3 (http://frodo.wi.mit.edu/). qRT-PCR was performed using an iCycler (Bio-Rad) with the threshold cycle number determined by use of iCycler software, version 3.0. Reactions were performed in triplicate and the threshold numbers were averaged. Results for the GPX7 gene were normalized to HPRT1 gene (forward 5′-TTGGAAAGGGTGTT TATTCCTCA-3′ and reverse 5′-TCCAGCAGGTCAGC AAAGAA-3′), which had minimal variation in all normal and tumor samples tested, and is therefore considered to be a reliable and stable reference gene for RT-PCR. Expression was calculated by use of the formula 2^(Rt–Et)/2^(Rn–En), as previously described [32 (link), 33 (link)]. For all of the primary gastric carcinoma samples, the gene was considered downregulated if the relative mRNA expression was ≤ 0.6, as compared to their matched normal samples.

The thermal cycling system iCycler together with the myIQ single Color Real-Time PCR Detection system (Biorad, CA, USA) were used for qPCR amplification and detection. The qPCR reactions were carried out in duplicates of 25-μl reaction mixtures in 96-well plates (iCycler, Biorad) sealed with optical adhesive covers (Microseal ‘B’ film, Biorad). Each reaction contained 250 nM of each primer, 12.5 μl of Perfecta SYBR Green Super mix of IQ (Quanta Biosciences, MD, USA) and 2.5 μl of template DNA (about 50 ng). Negative controls prepared by replacing template DNA with diethylpyrocarbonate (DEPC)-treated water, were included in each run to ensure the absence of DNA contaminants in the reagents. The concentration of primers, annealing temperature and template DNA concentrations had been optimized before the actual experiments as previously described [37 (link)]. The qPCR reactions were conducted as follows: initial denaturation at 95°C for 3 min followed by 50 cycles of 20 s at 95°C, 20 s at 60°C, and 72°C for 10 s. Fluorescence was measured at the end of each extension step at 72°C. The temperature was increased from 55°C to 95°C at a rate of 0.2°C per s to establish the melting curve. The threshold cycle values (C_t) were automatically determined by MyIQ Optical System software (version 2.0) (Biorad).

Total RNA was isolated using the RNeasy Mini kit (Qiagen, Germantown, MD) and cDNA synthesis was performed using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Primers specific for mouse and human genes were designed using the online software Primer 3 (http://frodo.wi.mit.edu/primer3/). The forward and reverse primers were designed to span two different exons for each gene (human: c-MYC and CCND1; mouse: c-Myc and Ccnd1), as previously described [7 ]. All primers were purchased from Integrated DNA Technologies (IDT, Coralville, IA). The qRT-PCR was performed using an iCycler (Bio-Rad), with the threshold cycle number determined by use of iCycler software version 3.0. Reactions were performed in triplicate and the threshold cycle numbers were averaged. The results of the genes were normalized to housekeeping genes, 18S for human and actin for mouse. Expression ratios were calculated according to the formula 2^(Rt–Et)/2^(Rn–En) [50 (link)] where Rt is the threshold cycle number for the reference gene observed in the test samples; Et is the threshold cycle number for the experimental gene observed in the test samples, Rn is the threshold cycle number for the reference gene observed in the reference samples, and En is the threshold cycle for the experimental gene observed in the reference samples. Rn and En values were calculated as an average of all reference samples.