Selected article for: "diluted cdna and real time"
Author: Selinger, Christian; Tisoncik-Go, Jennifer; Menachery, Vineet D; Agnihothram, Sudhakar; Law, G Lynn; Chang, Jean; Kelly, Sara M; Sova, Pavel; Baric, Ralph S; Katze, Michael G
Title: Cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct MERS-CoV isolates
  • Document date: 2014_12_22
    • ID: 0y3m47lh_30
    Snippet: RNAs were reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The resulting cDNA samples were diluted 50X and run on the 7900HT Fast Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master mix (Life Technologies) and 200 nM primers. Primer sets were designed using Primer3 [44] . The primer sequences for cellular gene targets and the strand-specific primers for quantifying viral genomic RNA are listed i.....
    Document: RNAs were reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The resulting cDNA samples were diluted 50X and run on the 7900HT Fast Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master mix (Life Technologies) and 200 nM primers. Primer sets were designed using Primer3 [44] . The primer sequences for cellular gene targets and the strand-specific primers for quantifying viral genomic RNA are listed in Additional file 1: Table S5 . For cellular gene mRNA quantification, relative gene expression in infected samples compared to that in mock-infected samples was calculated using the 2 −ΔΔCT method [45] . The RPL14 gene was selected as an internal control (calibrator) due to non-significant changes in RPL14 gene expression in MERS-CoV SA 1-and MERS-CoV Eng 1-infected Calu-3 2B4 cells, as determined by microarray analysis.

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