Selected article for: "Applied Biosystems real time pcr system and gene expression"

Author: McWhirter, Sarah M.; Barbalat, Roman; Monroe, Kathryn M.; Fontana, Mary F.; Hyodo, Mamoru; Joncker, Nathalie T.; Ishii, Ken J.; Akira, Shizuo; Colonna, Marco; Chen, Zhijian J.; Fitzgerald, Katherine A.; Hayakawa, Yoshihiro; Vance, Russell E.
Title: A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP
  • Document date: 2009_8_31
  • ID: 3b8b8p61_42
    Snippet: Quantitative PCR. Stimulated cells were overlayed with RNAlater (Applied Biosystems) and stored. RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's protocol, and was treated with RQ1 RNase-free DNase (Promega). 0.5 µg RNA was reverse transcribed with Superscript III (Invitrogen). Platinum Taq DNA polymerase (Invitrogen) and EvaGreen dye (Biotium) were used for quantitative PCR assays and analyzed with a real-time PCR .....
    Document: Quantitative PCR. Stimulated cells were overlayed with RNAlater (Applied Biosystems) and stored. RNA was isolated using the RNeasy kit (QIAGEN) according to the manufacturer's protocol, and was treated with RQ1 RNase-free DNase (Promega). 0.5 µg RNA was reverse transcribed with Superscript III (Invitrogen). Platinum Taq DNA polymerase (Invitrogen) and EvaGreen dye (Biotium) were used for quantitative PCR assays and analyzed with a real-time PCR system (StepOnePlus; Applied Biosystems). All gene expression values were normalized to Rps17 (mouse) or SP9 (human) levels for each sample. The following primer sequences were used: mouse Ifnb, (forward) 5-ATAAGCAGCTCCAGCTCCAA-3 and (reverse) 5-CTGTCTGCTGGTGGAGTTCA-3; mouse Ifn5, (forward) 5-TGACCTCAAAGCCTGTGTGATG-3 and (reverse) 5-AAG-TATTTCCTCACAGCCAGCAG-3; mouse Rps17, (forward) 5-CGCC-ATTATCCCCAGCAAG-3 and (reverse) 5-TGTCGGGATCCACC-T CAATG-3; human IFN, (forward) 5-AAACTCATGAGCAGTCT-GCA-3 and (reverse) 5-AGGAGATCTTCAGTTTCGGAGG-3; and human SP9, (forward) 5-ATCCGCCAGCGCCATA-3 and (reverse) 5-TCAATGTGCTTCTGGGAATCC-3.

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