Quantitative Real-Time PCR (qRT-PCR) is one of the most in vitro innovative methods in molecular biology research for analyzing nucleic acids (RNA and DNA). It uses fluorescent reporter molecular techniques to track production of amplified products and analyze expression of specific genes (decipher the functional genes) through creation of cDNA from RNA as in vivo (DNA Replication) depending upon exponential amplification of specific sequence between target and reference genes producing millions of copies for various analysis such as standard curve method, CT and 2−ΔΔCq methods to analyze gene expression during linear ground phase, exponential phase, log-linear phase and plateau phase of reaction cycles. Therefore, this review paper discusses the important and effectiveness of qRT-PCR in molecular biology researches. Thus, the essential prerequisites for accurate gene expression quantification are RNA extraction, RNA purification, cDNA synthesis, housekeeping genes as an internal control and qRT-PCR analysis to ascertain the amount of gene expression to investigate expression profiles of multigene families and endogenous genes by choosing appropriate reference genes as standard for normalizing target gene expression data to detect chromosomal translocation and single nucleotide polymorphism of relatively large number of samples in a short period of time. Hence, qRT-PCR use to understand basic cellular mechanisms and identifying changes in gene expression levels in respond to particular biological stimuli

qRt-PCR reaction, housekeeping genes, Normalization, CT method, 2−ΔΔCq method

qRT-PCR consider one of the most sensitive, effective, quick and reproducible technique for measuring gene expression profiles. Therefore, it preferred by many researches worldwide to achieve biological results by choosing suitable reference genes to guarantee accuracy data across molecular biology researches

Ammar M. F. Mohammed, Fatma A. Ali, Shahd A. Mohammed, Shahd M. Omran, Marwa M. Dosokey, Sayed Gebril, Tarek H.M. Elsharouny, Ahmed E. Mahmoud, Ismail M.A. Bedawy, E. Ismail and Haitham M.A. Elsayed (2025). qRT-PCR’s Significance and Efficacy in Molecular Biology Research. International Journal on Emerging Technologies, 16(2): 36–41.