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

Abstract: iNOS is inducible by a variety of factors related to inflammation and referred to as inducible NOS(iNOS). It is regulated at the level of gene expression; once expressed, it produces NO at a high rate. iNOS gene-expression profiling is an important tool in understanding molecular markers of the responses of cells and tissues to external factors. In this article a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) protocol was optimized to extract RNA (ribonucleic acid) from chicken spleen and to measure the expression levels of iNOS mRNAs from each sample. Detailed procedure was described for the analysis of iNOS levels. b-actin was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. Co-amplification of the iNOS gene with housekeeping gene (b-actin) provides a quantitative result. Changes in gene expression level may be monitored, while avoiding sample-to-sample loading variation. Key words: iNOS mRNA, RT-PCR, gene expression, b-actin.