Lightcycler faststart dna master sybr green 1

mRNA was extracted from in vitro PBMCs samples using the TRIzol Reagent (Molecular Research Center, Inc., Cincinnati, OH), according to the manufacturer’s instructions. cDNA was synthesized in a PerkinElmer Cetus DNA thermal cycler (PerkinElmer, Inc., Waltham, MA) using the SuperScript Reverse Transcription system (Takara, Shiga, Japan). A LightCycler 2.0 instrument (Roche Diagnostics; software version 4.0) was used for PCR amplification. All PCR reactions were performed using LightCycler FastStart DNA Master SYBR Green I (Takara), according to the manufacturer’s instructions. S2 Table shows the sense and antisense primers used for each molecule (5′→3′).

RNA was extracted using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized using the Superscript Reverse Transcription System (TaKaRa, Shiga, Japan), and quantitative real-time polymerase chain reaction (PCR) was performed using LightCycler FastStart DNA Master SYBR Green I (TaKaRa) according to the manufacturer’s instructions. The primer sequences for PCR were designed using Primer Express (Applied Biosystems, Foster City, CA, USA) and were as follows: ndufb5 (forward: TCC CAG AAG GCT ACA TCC CT, reverse: ATT CCG GGC GAT CCA TCT TG), uqcrb (forward: TCA AGC AAG TGG CTG GAT GG, reverse: TCA GGT CCA GGG CTC TCT TA), cox5b (forward: ATG GGT CCA GTC CCT TCT GT, reverse: GCT TCA AGG TTA CTT CGC GG), cycs (forward: AAT CTC CAC GGT CTG TTC GG, reverse: GGT CTG CCC TTT CTC CCT TC), and β-actin (forward: GAA ATC GTG CGT GAC ATC AAA G, reverse: TGT AGT TTC ATG GAT GCC ACA G). The mRNA levels were normalized relative to that of β-actin.

Messenger RNA (mRNA) was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized using the SuperScript reverse transcription system (TaKaRa, Otsu, Japan). A LightCycler 2.0 instrument (software version 4.0; Roche Diagnostics, Basel, Switzerland) was used for polymerase chain reaction amplification. All reactions were performed using LightCycler FastStart DNA Master SYBR Green I (TaKaRa) according to the manufacturer’s instructions.
The following primers were used: IL-6, 5′-AAC GAT GAT GCA CTT GCA GAA A-3′ (sense) and 5′-TCT GAA GGA CTC TGG CTT TGT C-3′ (antisense); TNF-α, 5′-ATG AGC ACA GAA AGC ATG ATC-3′ (sense) and 5′-TAC AGG CTT GTC ACT CGA ATT-3′ (antisense); IL-17, 5′-CCTCAAAGCTCAGCGTGTCC-3′ (sense), 5′-GAGCT CACTTTTGCGCCAAG-3′ (antisense); STAT3, 5′-CCG TCT GGA AAA CTG GAT AAC TTC-3′ (sense), 5′-CCT TGT AGG ACA CTT TCT GCT GC-3′ (antisense); and β-actin, 5′-GTA CGA CCA GAG GCA TAC AGG-3′ (sense) and 5′-GAT GAC GAT ATC GCT GCG CTG-3′ (antisense). All expression values were normalized by the amount of β-actin cDNA amplified from the same RNA sample and calculated by using the comparative delta-delta Ct method.

Messenger RNA (mRNA) was extracted using TRIzol (Molecular Research Center, Cincinnati, OH, USA). cDNA was synthesized using the Superscript Reverse Transcription system (Takara, Shiga, Japan), then qPCR was performed using LightCycler FastStart DNA Master SYBR green I (Takara) following the manufacturer's instructions. All expression values were normalized to that of GAPDH mRNA. The primer sequences are described in Table 1.

Total RNA was extracted using TRIzol (Molecular Research Center, Cincinnati, OH, USA), and 2 μg total RNA were reverse transcribed using the Superscript Reverse Transcription system (Takara, Shiga, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using Light-Cycler FastStart DNA master SYBR green I (Takara) fluorescent dye on the ABI PCR system. Primers targeting TGF-β (forward: 5′- TGC GGC AGC TGT ACA TTG A -3′, reverse: 5′- TGG TTG TAC AGG GCC AGG A -3′), α-SMA (forward: 5′- TGG GTG ACG AAG CAC AGA GC -3′, reverse: 5′- CTT CAG GGG CAA CAC GAA GC -3′), collagen-1 (forward: 5′- GTC ACC CAC CGA CCA AGA AAC C -3′, reverse: 5′- AAG TCC AGG CTG TCC AGG GAT G -3′), and β-actin (forward: 5′- GGA CTT CGA GCA AGA GAT GG -3′, reverse: 5′- TGT GTT GGG GTA CAG GTC TTT G -3′) were designed using Primer Express (Applied Biosystems, Foster City, CA, USA). The mRNA expression level of each gene was normalized to that of β-actin.

Total RNA was isolated from human chondrocytes using the TRIzol method (Invitrogen). Complementary DNA (cDNA) was prepared by reverse transcription of single-stranded RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer's instructions. mRNA expression was estimated using real-time quantitative PCR with LightCycler FastStart DNA Master SYBR green I (Takara), according to the manufacturer's instructions. The primer pairs used in these reactions were as follows: for control human β-actin, forward 5′-GGA CTT CGA GCA AGA GAT GG-3′, reverse 5′-TGT GTT GGC GTA CAG GTC TTT G-3′; for human tissue inhibitor of metalloproteinase (TIMP)-1, forward 5′-AAT TCC GAC CTC GTC ATC AG-3′, reverse 5′-TGC AGT TTT CCA GCA ATG AG-3′; for human ADAMTS5, forward 5′-TAT GAC AAG TGC GGA GTA TG-3′, reverse 5′-TTC AGG GCT AAA TAG GCA GT-3′. The amplification reactions, data acquisition, and analysis were performed with the LightCycler Real-Time PCR system (Roche Diagnostics, Indianapolis, IN, USA), and the relative levels of gene expression were normalized against β-actin.

PCR amplification and analyses were performed using a LightCycler 2.0 instrument (Roche Diagnostics, Mannheim, Germany) with software version 4.0. All reactions were performed using LightCycler Fast Start DNA Master SYBR green I (Takara, Shiga, Japan), according to the manufacturer’s instructions. The following primers were used: TRAP, 5′-TCC TGG CTC AAA AAG CAG TT-3′ (sense) and 5′-ACA TAG CCC ACA CCG TTC TC-3′ (antisense); calcitonin receptor, 5′-CGG ACT TTG ACA CAG AA-3′ (sense) and 5′-AGC AAT CGA CAA GGA GT-3′ (antisense); integrin b3, 5′-CTG TGG GCT TTA AGG ACA GC-3′ (sense) and 5′-GAG GGT CGG TAA TCC TC-3′ (antisense); cathepsin K, 5′-CAG AGG TGT GTA CTA TG-3′ (sense) and 5′-GCG TTG TTC TTA TTC CGA GC-3′ (antisense); RIPK1, 5′-CTG TTC CCT GTG CCC AAT AA-3′ (sense) and 5′-ATG ACT CTG AAG CTG TCC TTT C-3′ (antisense) and RIPK3, 5′-GCA CTC CTC AGA TTC CAC ATA C-3′ (sense) and 5′-GTG TCT TCC ATC TCC CTG ATT C-3′ (antisense).