Mirna extraction kit

Cell transfections with HMGB1 siRNA and miR-200c mimic and inhibitor were performed as described previously [24 (link)]. A549 cells were transfected with HMGB1 siRNA (AM16106; Life Technologies), miR-200c-3p mimic (MC11714; Life Technologies) or miR-200c inhibitor (MH11714; Life Technologies)using the Lipofectamine^® RNAiMAX kit (Thermo Fisher Scientific). Transfection mixtures containing 0.25 ml of Lipofectamine^® 2000 (Life Technologies), 25 ml of Opti-MEM^™ (Life Technologies), and 10 nM siRNA or15 pmol/ml miR-200c inhibitor or mimic were incubated at room temperature for 10 min and then added to A549 cells seeded in 6-well plates in medium containing 10% (v/v) FBS. Cells were harvested after 48 h and analysed for miRNA expression using a miRNA extraction kit (Life Technologies) and quantitative RT-PCR.

Quantitative real-time PCR was performed as described previously [31 (link)]. Total RNA (2 μg) was reverse-transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and miRNA was extracted using a miRNA extraction kit (Life Technologies, Carlsbad, CA, USA). The resultant cDNA diluted 1:10 was used as a template to quantify the relative content of mRNA by real-time TaqMan PCR (LightCycler FastStart DNA Master SYBR Green I, Roche, Indianapolis, IN, USA); cDNA diluted 1:5 was used as a standard. The following primers obtained from Integrated DNA Technologies (MDBio, Taipei, Taiwan) were used: LIN28A forward: 5′-CAA AAG GAA AGA GCA TGC AGA A-3′, reverse: 5′-ATG ATC TAG ACC TCC ACA GTT GTA GC-3′; MMP2 forward: 5′-TCC AAC CAC CAC CAC AC-3′, reverse: 5′-AGT CCA AAG AAC TTC TGC AT-3′; MMP9 forward: 5′-TCC AAC CAC CAC CAC AC-3′, reverse: 5′-CGG ACT CAA AGG CAC AGT A-3′; GAPDH forward: 5′-AGC CAC ATC GCT CAG ACA-3′, reverse: 5′-GCC CAA TAC GAC CAA ATC C-3′. U6 (P01183321) primer and miRNA-98 (P01495350) were obtained from Life Science Technology (Gaithersburg, MD, USA). Relative mRNA expression was calculated by normalizing target mRNA levels to those of house-keeping genes (GAPDH and U6) and compared by the CT (ΔΔCT) method.

Quantitative real-time PCR was performed as described previously [20 (link)]. Total RNA (2 μg) was reverse-transcribed using the SuperScript^® First-Strand Synthesis System for real time PCR (Invitrogen), and miRNA was extracted using a miRNA extraction kit (Life Technologies). The resultant cDNA, diluted 1:10, was used as a template to quantify the relative content of miRNA by real-time TaqMan^® PCR (LightCycler^® FastStart DNA Master SYBR^® Green I, Roche, Basel, Switzerland), and cDNA diluted 1:5 was used as a standard. The following primers obtained from Integrated DNA Technologies (Coralville, IA, USA) were used: HMGB1 forward: 5′- GTT CAA GGA TCC CAA TGC AC -3′, reverse: 5′- GAT TTT TGG GCG ATA CTC AGA -3′; CTNNB forward: 5′- CCA GCT CTC TCT TCA GAA CAG -3′, reverse: 5′- GGG TCC ATA CCC AAG GC -3′; ACTA2 forward: 5′- GAC AAT GGC TCT GGG CTC TGT AA -3′, reverse: 5′- ATG CCA TGT TCT ATC GGG TAC TT 3′; GAPDH forward: 5′-AGC CAC ATC GCT CAG ACA-3′, reverse: 5′-GCC CAA TAC GAC CAA ATC C-3′. Primer pairs for the amplification of U6 (P01183321) and miR-200c (P01420724) were obtained from Life Science Technology (MDBio). Relative mRNA expression was calculated by normalising the target mRNA levels to those of house-keeping genes (GAPDH and U6) and the data were compared among treatment groups by the ^ΔΔCT method.