Quantitative RT-PCR for Interferon and Chemokine

RNA isolation was performed by using an RNeasy minikit according to the manufacturer's instructions (Qiagen, CA). Total RNA was treated with Turbo DNase (Ambion, TX) to remove residual genomic DNA contamination. First-strand cDNA synthesis was carried out by using Moloney murine leukemia virus reverse transcriptase (MMLV-RT) (Promega, WI) on 2 μg of total RNA according to the manufacturer's instructions. SYBR green PCR master mix (Applied Biosystems, CA) was used along with previously described gene-specific primers for Ifnα, Ifnβ, or Ccl5 to detect the presence of an amplified product (9 (link)). All samples were run in duplicate. Results were analyzed by using relative quantification with 7300 SDS software (Applied Biosystems, CA). Data were normalized to the values for the mouse gene Ywhaz, which is constitutively expressed with minimal change (9 (link), 47 (link)).