For arthritis analysis, RNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as previously described [21 (link)]. Briefly, total RNA was extracted from four amputated limbs using a RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR was performed using an Applied Biosystem StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with Taqman probes and the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), and Actb (Mm00607939_s1).
For brain analysis, RNA extraction and RT-PCR were performed as follows. After transcardial perfusion with PBS, total RNA was extracted from the dissected medulla containing of the AP (from the rostral to caudal end) using a RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA extraction from the whole brain after separating the medulla oblongata was similarly performed. RT-PCR was performed as described above with the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), TGF-β (Mm01178820_m1), Itgam (Mm00434455_m1), and Gapdh (Mm99999915_ g1).
Expression levels normalized to Actb or Gapdh were analyzed using the ΔΔCT method. mRNA expression levels were represented as values relative to the average of the saline group.
For brain analysis, RNA extraction and RT-PCR were performed as follows. After transcardial perfusion with PBS, total RNA was extracted from the dissected medulla containing of the AP (from the rostral to caudal end) using a RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA extraction from the whole brain after separating the medulla oblongata was similarly performed. RT-PCR was performed as described above with the following primers: IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), TGF-β (Mm01178820_m1), Itgam (Mm00434455_m1), and Gapdh (Mm99999915_ g1).
Expression levels normalized to Actb or Gapdh were analyzed using the ΔΔCT method. mRNA expression levels were represented as values relative to the average of the saline group.
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