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.