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Unformatted text preview: onal, Camarilo,
CA, USA). The minimal detectable doses of IL-6 and
TNF-α were 2 pg/ml and 0.5 pg/ml, respectively.
Reverse transcriptase–polymerase chain reaction
Total RNA was extracted from retrieved tissues or
cultured osteoblasts using Easy Prep RNA (Takara
Biochemicals, Tokyo, Japan). Reverse transcriptase–
polymerase chain reaction (RT-PCR) was performed as follows using a Onestep RT-PCR kit (QIAGEN,
Tokyo, Japan). Brieﬂy, total RNA (0.1 µg) was reversetranscribed into cDNA and subjected to PCR. After
initial denaturation at 95°C for 15 min, PCR for IL-6,
TNF-α, osteoclast differentiation factor (ODF), osteoprotegerin (OPG), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed for 35 cycles at
94°C for 1 min, 62°C for 1 min, and 72°C for 1 min. PCR
for collagen I was performed for 30 cycles at 95°C for
30 s, at 60°C for 30 s, and at 60°C for 30 s. PCR for matrix
metalloproteinase (MMP)-13 was performed for 35
cycles at 90°C for 1 min, at 54°C for 2 min, and at 72...
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- Spring '08