Quantitative PCR Protocol for Gene Expression

After validating RNA purity and quality, cDNA was subsequently synthesized, and RT-PCR was performed using a commonly used housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a positive control. Synthesis of cDNA was performed using an Affinity Script QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) with a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems, Waltham, MA, USA) under conditions of 25 °C for 5 min, 42 °C for 5 min, 55 °C for 40 min, and 95 °C for 5 min. In addition, cDNA was amplified in solutions containing EmeraldAmp PCR Master Mix (Takara, Tokyo, Japan) and the relevant primers. The 3′-UTR gene-specific primers were targeted against the genes listed in Table 1. Sequences for MKP-1, CEBPD, KLF4, LEF1, SUMO1/SENP5, MMP-8, KSR1, and NGFR were obtained from the National Center for Biotechnology Information (NCBI) database. Thermal cycler parameters for PCR were as follows. After initial denaturation at 97 °C for 5 min, 25–35 cycles of 95 °C for 45 s, 55–65 °C for 45 s, and 72 °C for 1 min were performed. A final elongation step was performed at 72 °C for 10 min. PCR reaction solutions were then loaded onto a 1.6% agarose gel. Electrophoresis (100 V, 31 min or 85 V, 39 min) was performed in 1× TAE buffer, using a Mupid-ex electrophoresis system (Advance, Tokyo, Japan). Gels were stained with ethidium bromide for 10 min. Bands were then visualized using a ChemiDoc XRS Plus imaging system and quantified using Image Lab Software, version 3.0 (Bio-Rad). The intensity of all samples obtained was expressed relative to GAPDH expression in each sample. All assays were performed in duplicate, and the mean value was used as the value for one individual. An experimental series was performed twice using samples from different rats. When the second series was assayed, several samples measured in the first series were included to correct the absolute counts. Such measurements were repeated 3 times to ensure reproducibility. In preliminary experiments, we first clarified the PCR cycle number at which the expression level of each gene saturates. Based on these results, we adopted the PCR cycle number lower than the gene expression level saturating in the present study. We also performed RT-PCR and electrophoresis in a dilution series of the amount of cDNA in a sample containing a high amount of each gene and confirmed that the relationship between the amount of cDNA and its OD was linear. The intensities obtained from each sample were within the linear range in the preliminary experiments described above.