The reverse transcription polymerase chain reaction (RT-PCR) is one of the most sensitive methods for the detection and quantitation of mRNA. It is widely used for quantification of mRNA levels and is useful tool for basic research, agriculture, medicine and biotechnology. Introduction of Real-time technique significantly improves rapidity, sensitivity, specificity and reproducibility of RT-PCR method. Real-time PCR detects and quantifies nucleic acids even from live and dead pathogens or cells. This review discusses the background, advantages and limitations of conventional and Real-time RT-PCR methods for quantitation of gene expression and its application in basic, agricultural and biomedical research.