Iq5 multi color real time pcr

Mice bearing implants were lavaged with 2 ml PBS at 2, 4 and 7 d post implantation and the implants and peritoneal lavage fluid were collected. Cell isolates from lavage samples were prepared as described previously [26 (link)]. RNA was extracted from implants or lavage cell isolates with the RNeasy Mini Kit from Qiagen (Valecia, CA). 1μg of total RNA per sample was translated into single-stranded cDNA using the Superscript II cDNA synthesis kit and Oligo-dT both from Invitrogen (Carlsbad, CA), according to supplier’s instructions. Gene expression levels were determined using the IQ5 multi color real-time PCR and SYBR green supermix from Bio-Rad Laboratories, Inc. (Hercules, CA). The amplification of each sample was performed in duplicate. Data was normalized to the levels of the housekeeping gene GAPDH and displayed normal distribution. Mouse-specific primers used to amplify mRNA sequences were:
Fold-change was determined in comparison to WT samples at 24 h. A total of five mice per time point per genotype were analyzed and the experiment was performed twice.

miRNA or mRNA was extracted from Millipore filter implants in vivo or BMDM plated on petri plastic in vitro with the miRNeasy or mRNeasy Mini Kit from Qiagen (Valecia, CA). 1μg of total RNA or 2μg miRNA per sample was translated into single-stranded cDNA using the Superscript II cDNA synthesis kit and Oligo-dT both from Invitrogen (Carlsbad, CA), according to supplier’s instructions for mRNA, or using miScript II RT Kit from Qiagen (Valecia, CA). Gene expression levels were determined using the IQ5 multi-color real-time PCR and SYBR green supermix from Bio-Rad Laboratories, Inc. (Hercules, CA) for mRNA and miScript SYBR Green qPCR kit from Qiagen (Valecia, CA). The amplification of each sample was performed in triplicate. For mRNA, data was normalized to the levels of the housekeeping gene β-actin and displayed normal distribution. For miRNA samples, primers from Qiagen were used: mmu-miR-223-3p, mmu-miR-194-3p, and hs-RNU6-2 primer assays were used. Fold-change was determined in comparison to non-IL4-treated WT samples at 24 h.