ABSTRACT
ln recent years, real-time PCR has come forward as a robust and widely used molecular technique in clinical and biological. Although it can detect very minute quantities of target nucleic acid, but quantification of specific nucleic acids is not an easy task. Accurate and precise quantification is hampered by a number of factors that may include assay development and validation, fluorophores selection, handling during sample preparation, storage, reaction procedures, and batch analysis conditions. Even minor variations are significantly magnified by the exponential nature of this technique. Current review gives an insight of the advantages, limitations, assay chemistries, quantitation parameters, and quality control issues related to this technology. Moreover it will also highlight the utilization of Real time PCR in clinical oncology, virology, microbiology, and gene expression studies.