Rotor gene 6000 real time pcr machine

Quantitative PCR was performed in triplicates using a SYBR Green kit SensiFAST SYBR No-ROX (Bioline, Sydney, Australia) with Rotorgene 6000 real-time PCR machine (Corbett Research, Sydney, Australia). The PCR reaction was performed in a volume of 10 μL containing 5 μL of 2 × SensiFAST, 400 mM of each primer and 2 μL of diluted cDNA template. Post thermal cycling, amplification cycle (Cq) values for all genes were collected and imported into qBase+ version 3.0 (Biogazelle, Zwijnbeke, Belgium) software and analyzed against 2 optimized reference genes, GAPDH and HMBS, in the present study. The qBase+ applied an arithmetic mean method to transform logarithmic Cq value to linear relative quantity using exponential function for relative quantification of genes (Vandesompele et al., 2002 (link); Hellemans et al., 2007 (link)) and the output data were exported to a statistical software for further analysis.

Real-time quantitative PCR was performed using a Rotorgene 6000 real-time PCR machine (Corbett Research, Sydney, Australia). The qBase + version 3.0 (Biogazelle, Zwijnbeke, Belgium) software was employed to determine the 2 most stable genes among 7 (18S, β-actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], hypoxanthine phosphoribosyltransferase 1 [HPRT1], hydroxymethylbilane synthase [HMBS], TATA-box binding protein [TBP] and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation [YWHAZ]) different reference genes. Based on the expression stability HPRT1 and TBP were used to normalize the target genes in the jejunum. The target genes (Table 1) determined were: claudin 1 (CLDN1), tight junction protein 1 (TJP1), junctional adhesion 2 (JAM2), occludin (OCLD), mucin 2 (MUC-2), mucin 5 (MUC5AC), aminopeptidase N (APN), calbindin 1 (CALB1), ATPase Na⁺/K⁺ transporting subunit alpha 1 (ATP1A1), ATP synthase subunit alpha (ATP5A1W), calcium-sensing receptor (CaSR), calcium channel, voltage-dependent, P/Q type alpha 1A subunit (CACNA1A), Na-dependent Pi cotransporters, type IIb (NaPi-IIb) and vitamin D receptor (VDR). The relative expression of genes using the arithmetic mean method in qBase+ was exported to SAS 9.3 package for further analysis.

Quantitative PCR was performed using a Rotorgene 6,000 real-time PCR machine (Corbett Research, Sydney, Australia). The qBase + version 3.0 (Biogazelle, Zwijnbeke, Belgium) software was used to determine the 2 most stable genes among seven (18S, ACTB, GAPDH, HPRT1, HMBS, TBP, and YWHAZ) different reference genes. Based on the expression stability, GAPDH and HPRT1 were used to normalize the target genes in the jejunum. The relative quantification of genes using the arithmetic mean method in qBase+ was exported to SAS 9.3 package for further analysis.

Total RNA was extracted from each sample using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Quality of the RNA was evaluated using the NanoDrop 1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Inc.) and first-strand cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara, Bio, Inc., Otsu, Japan). For miR-23b, the reverse transcription primer was used. SYBR Green II dye-based qPCR analysis was conducted using the Rotor-Gene 6000 Real-Time PCR machine (Corbett Research, Mortlake, Australia). The amplification program was set as follows: Initial denaturation at 94°C for 10 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 15 sec and extension at 70°C for 15 sec; and a final extension step at 70°C for 5 min. The RT-qPCR was conducted using the 2^−∆∆Ct method and each sample of 0.1 µg of cDNA was tested in triplicate (13 (link)). Glyceraldehyde 3-phosphate dehydrogenase and U6 were used as endogenous normalization controls. The primer sequences of the genes are presented in Table I.