Journal: American Journal of Transplantation
Article Title: Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival, et al. Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival
Figure Lengend Snippet: RT q PCR analysis showing relative gene expression levels of IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, TNF ‐α, and IFN ‐γ in allograft skin (A – H) and muscle (I – P). For RT q PCR , a total volume of 15 μl per reaction was used containing 50 ng cDNA template, 1.5 μl QuantiTect Primer Assay (Qiagen), 7.5 μl QuantiTect SYBR green PCR kit (Qiagen), and 5 μl ddH2O. Primers for IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, IFN ‐γ, and TNF ‐α were obtained from Qiagen. Cycling conditions included a hot start activation (5 minutes, 95°C), 40 cycles of 10 seconds denaturation (95°C), and annealing and extension (30 seconds, 60°C). Amplicons were quantified with the comparative threshold cycle ( CT ) method; data acquisition was performed using the 7500 System SDS Software Version 2.0.5 (Applied Biosystems). Amplification specificity was checked using melting curve according to the manufacturer's instructions. All samples were measured in duplicate and respective results were averaged. A–P, In allograft skin (A–H) and muscle (I–P) no significant differences in relative gene expression of cytokines was observed between anti‐ IL ‐1β‐treatment groups 3 and 4 and group 2 controls. Expression levels of all markers were normalized to those of group 2
Article Snippet: One micrograms of total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen).
Techniques: Polymerase Chain Reaction, Expressing, SYBR Green Assay, Activation Assay, Software, Amplification