Quantitative PCR Gene Expression Profiling

The quantitative PCR method can obtain expression profiles of genes of interest in a high-throughput and accurate manner^{22 (link)}. In this work, the same experimental conditions were used⁴⁴ . RNA isolation was based on the suggested protocol in the TRI Reagent-RNA Kit (Molecular Research Center, Cincinnati, OH, USA). RNA samples for quantitative PCR were pre-treated with DNase I (Promega, Madison, WI, USA). The DNA primers used in the quantitative PCR were designed by Primer Express software (Applied Biosystems, Foster City, CA, USA) and their complements (Table S5). DNA was synthesised using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The quantitative PCR experiments were conducted in an ABI PRISM® 7000 instrument (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq reagent (Takara, Tokyo, Japan). All quantitative PCR experiments were performed with two replicates.