Abi 7300 sequence detector

Total RNA from liver tissues and cells was extracted using TRIzol (Takara, China) and reverse-transcribed using the PrimeScript™ RT reagent kit (Takara, China) according to the manufacturers' instructions. Quantitative PCR was carried out on the ABI 7300 sequence detector (Applied Biosystems, Rotkreuz, Switzerland). The primer sequences used are summarized in Table 1. Gene expression values were calculated based on −△Ct method and normalized to expression of GAPDH. Results were calculated as 2^−△△Ct and express the x-fold increase of gene expression compared to control rats or cells.

RNA was extracted from HCT116 or SW480 cells treated with 2 mM NaB for 24 h with TRIzol reagent (Life Technologies) by the manufacturer's protocol. Reverse transcription was accomplished with the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). Real-time PCR was performed using SYBR green reagents (Takara Bio) in an ABI 7300 sequence detector (Applied Biosystems, Foster City, CA, USA) to determine the relative expression of genes of interest. The sequences of the PCR primers used are present in Supplementary Table S1. The 18S gene served as the internal control.

Total RNA of the colon, liver and epididymal fat was isolated using TRIzol (Invitrogen, China), and 1 μg RNA was reverse transcribed with standard reagents (Biocolors, China). The reaction system was 10 μl including 5 μl SYBR, 1 μl DNA (100 ng μl⁻¹), 0.5 μl forward and reserve primers (10 mM μl⁻¹) and 3 μl double distilled water. The expression of PPARγ, LPL, Fiaf, PP2A, SREBP-1c, ChREBP, FASN, leptin and adiponectin mRNA was measured by quantitative real-time PCR with SybrGreen (Roche, Switzerland), and fluorescence was detected on an Applied Biosystems (ABI) 7300 sequence detector (He et al., 2004 (link); Cong et al., 2007 (link); Jun et al., 2008 (link)). Samples were incubated in the ABI 7300 sequence detector for an initial denaturation at 95°C for 10 min, followed by 35 PCR cycles of 95°C for 15 s, 60°C for 1 min and 72°C for 1 min. The expression of the genes was calculated relative to the expression of GAPDH, β-actin and HPRT with formula 2^-ΔΔCt. Amplification of specific transcripts was confirmed by melting curve profiles at the end of each PCR.

Based upon information from the recent genetic association studies, two SNPs, that is, rs13266634(Arg276Trp C/T) and rs11558471(A/G) in the SLC30A8 gene, were selected for our study [4 (link)-9 (link)]. The first SNP is non-synonymous, in which the amino acid arginine is changed to tryptophan, while the second one is a synonymous SNP at the 3′-UTR. The SLC30A8 gene structure and location of these two polymorphisms are shown in Figure 1. Genotyping these two SNPs were done using TaqMan SNP genotyping assays purchased from Applied Biosystems (Applied Biosystems, Foster City, USA). Genotyping experiments were performed in ABI 7300 sequence detector with a Taqman allelic discrimination protocol (Applied Biosystems). DNA samples were distributed randomly across plates with cases and controls for genotyping quality control. All PCR reactions were run in 20 μl volumes using 10 to 20 ng genomic DNA. Millipore water was used as negative controls (blanks) on each plate.

RNA from tissues was isolated using TRIzol reagent (Takara Bio, Japan) and reverse transcribed using PrimeScript RT Master Mix for RT-PCR (TaKaRa Bio, Japan). Real-time PCR was performed using SYBR Select Master Mix (Applied Biosystems, USA) on an ABI 7300 sequence detector (Applied Biosystems, USA). The results were normalised to the 18S level in each sample, and endogenous U6 small nuclear RNA or cel-miR-39-3p were used for normalisation of cellular or plasma miRNA. All reactions were carried out in triplicate, and analysis was carried out using the 2^−ΔΔCt method. A complete list of PCR primers is shown in Supplementary Table 2.