Semiquantitative RT-PCR analysis to assess the expressionlevels of iNOS in chicken macrophages

iNOS is inducible by a variety of factors related to inflammation and referred to asinducible NOS(iNOS). It is regulated at the level of gene expression; once expressed, it producesNO at a high rate. iNOS gene-expression profiling is an important tool in understandingmolecular markers of the responses of cells and tissues to external factors. In this article asemiquantitative reverse transcription-polymerase chain reaction (RT-PCR) protocol wasoptimized to extract RNA (ribonucleic acid) from chicken spleen and to measure the expressionlevels of iNOS mRNAs from each sample. Detailed procedure was described for the analysis ofiNOS levels. β-actin was used as an internal control to normalize for sample to sample variationsin total RNA amounts and for reaction efficiency. Co-amplification of the iNOS gene withhousekeeping gene (β-actin) provides a quantitative result. Changes in gene expression levelmay be monitored, while avoiding sample-to-sample loading variation.