2.4 LightCycler real-time PCR
The cDNA levels of IL-4 was quantitated by real-time PCR using the Quantitect™ SYBR green PCR Kit (Takara, Japan) and a LightCycler® 2.0 (Roche Molecular Biochemicals, Switzerland). The primers of IL-4 used for RT-PCR were as follows: 5′-TCATTTTCCCTCGGTTTCAG-3′(forward) and 5′- ATAGGTGTCGATTTGCAGTG-3′ (reverse). The cDNA levels of miR-24 and miR-27 were quantitated by real-time PCR using the miScript SYBR Green PCR Kit (Qiagen) and the LightCycler® 2.0. The primers used for miRNA real-time PCR were designed according to the recommendations for use with the miRNA miScript Primer Assay (Qiagen). The second derivative maximum method was performed for CP determination using LightCycler software V3.5.30 (Roche Molecular Biochemicals). After normalization with Relative Quantification Software V1.0 (Roche Molecular Biochemicals), the final results were calculated as ratios of the target genes or miRNAs to the amount of GAPDH or U6.