Real quant software

Total RNA was isolated from siRNA-transfected cells at different time points with the TRIzol Reagent (Invitrogen). RNA samples (2.5 to 5 μg) were subjected to reverse transcription (RT) using the Omniscript reverse transcriptase kit (Qiagen) and amounts of HDAC1 and β-actin (internal control) were subsequently quantified by real-time PCR, using the SYBR Green Master Mix (Takara Bio, Inc.). PCR was conducted with 1.25–2.5 ng of cDNA along with primers specific for HDAC1 (0.5 μM) or β-actin (0.25 μM). The primer pairs were: HDAC1-sense 5′-TCCGAGACGGGATTGATGACG-3′ and HDAC1-antisense 5′-CCCAGCATCAGCATAGGCAGG-3′, β-actin sense 5′-GGGTCAGAAGGATTCCTATG-3′ and β-actin antisense 5′-GGTCTCAAACATGATCTGGG-3′. The cycling conditions were: initial denaturation at 95 °C for 10 s followed by 50 cycles of amplification (denaturation at 95 °C for 5 s, hybridization at 60–65 °C for 20 s and elongation at 72 °C for 15 s). Data were analyzed with the Real Quant Software (Roche Applied Science). Endogenous HDAC1 protein levels in siRNA-transfected cells were analyzed by Western blot.

Total RNA was extracted from 100 mg tissues or 1 × 10⁵ cells using the RNeasy RNA Mini Kit (Qiagen). First strand cDNA was synthesized using POWERSCRIPT reverse transcriptase (Clontech). The following gene-specific primer pairs were used for quantitative PCR:

BRG1: Forward, 5′- TCATGTTGGCGAGCTATTTCC -3′;

Reverse, 5′- GGTTCCGAAGTCTCAACGATG-3′.

MMP2: Forward, 5′- TTACTTGTGGAGCCGCTGAC -3′;

Reverse, 5′- TCAGATGGTGCCAGCAATAG -3′.

MMP9: Forward, 5′- GCTATTTCGGCATGTTGATCC -3′;

Reverse, 5′- GAAGTTAACCTCGGATCCTGG-3′.

GAPDH: Forward, 5′- GCTGAGTATGTCGTGGAGTC -3′;

Reverse, 5′- AGTTGGTGGTGCAGGATGC -3′.

PCR was performed using a Fast Start Master SYBR Green Kit (Roche) on a LightCycler (Roche). The expression level of target gene mRNA was analyzed using RealQuant software (Roche) and normalized to that of GAPDH mRNA.