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 Ifnî¡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 IFNî¢, (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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