The What, How and Why of qPCR Analysis

References 1. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990;262(4):56–65. doi: 10.1038/scientificamerican0490-56 2. Kaltenboeck B, Wang C. Advances in real-time PCR: application to clinical laboratory diagnostics. Adv Clin Chem. 2005;40:219–259. doi: 10.1016/s0065-2423(05)40006-2 3. McDonald C, Taylor D, Linacre A. PCR in forensic science: a critical review. Genes. 2024;15(4):438. doi: 10.3390/genes15040438 4. Rahman MT, Uddin MS, Sultana R, Moue A, Setu M. Polymerase chain reaction (PCR): a short review. Anwer Khan Mod Mel Col J. 2013;4(1):30–36. doi: 10.3329/akmmcj.v4i1.13682 5. Adams G. A beginner’s guide to RT-PCR, qPCR and RT-qPCR. Biochem (Lond). 2020;42(3):48–53. doi: 10.1042/BIO20200034 6. Steward K. Agarose gel electrophoresis, how it works and its uses. Technology Networks. https://www.technologynetworks.com/genomics/articles/agarose-gel-electrophoresis-how-itworks-and-its-uses-358161. Updated February 2, 2024. Accessed August 20, 2025. 7. Alvarez-Garcia V, Bartos C, Keraite I, et al. A simple and robust real-time qPCR method for the detection of PIK3CA mutations. Sci Rep. 2018;8:4290. doi: 10.1038/s41598-018-22473-9 8. Klymus KE, Ruiz‑Ramos DV, Thompson NL, Richter CA. Development and testing of species‑specific quantitative PCR assays for environmental DNA applications. J Vis Exp. 2020;(165):e61825. doi: 10.3791/61825 9. Chapela M-J, Garrido-Maestu A, Cabado AG. Detection of foodborne pathogens by qPCR: a practical approach for food industry applications. Cogent Food Agric. 2015;1(1):1013771. doi: 10.1080/23311932.2015.1013771 10. Kunz A, Gern U, Schmitt A, et al. Optimized assessment of qPCR-based vector copy numbers as a safety parameter for GMP-grade CAR T cells and monitoring of frequency in patients. Mol Ther Methods Clin Dev. 2020;17:448–454. doi: 10.1016/j.omtm.2020.02.003 11. Bruckert G, Vivien D, Docagne F, Roussel BD. Normalization of reverse transcription quantitative PCR data during ageing in distinct cerebral structures. Mol Neurobiol. 2016;53(3):1540– 1550. doi: 10.1007/s12035-015-9114-5 12. D’haene B, Vandesompele J, Hellemans J. Accurate and objective copy number profiling using real-time quantitative PCR. Methods. 2010;50(4):262–270. doi: 10.1016/j. ymeth.2009.12.007 13. Ruijter JM, Pfaffl MW, Zhao S, et al. Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications. Methods. 2013;59(1):32–46. doi: 10.1016/j.ymeth.2012.08.011 14. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008;44(5):619–626. doi: 10.2144/000112776 15. Altar CA, Vawter MP, Ginsberg SD. Target identification for CNS diseases by transcriptional profiling. Neuropsychopharmacology. 2009;34(1):18–54. doi: 10.1038/npp.2008.172 16. Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 2009;67(1):6–20. doi: 10.1111/j.1574- 6941.2008.00629.x 17. Zhang H, Yan Z, Wang X, et al. Determination of advantages and limitations of qPCR duplexing in a single fluorescent channel. ACS Omega. 2021;6(34):22292–22300. doi: 10.1021/ acsomega.1c02971 18. Is qPCR more accurate than PCR? PHC Holdings Corporation. https://www.phchd.com/eu/biomedical/knowledge-base/is-qpcr-more-accurate-than-pcr. Published August 19, 2021. Accessed August 6, 2025. The polymerase chain reaction (PCR) is a technique used to amplify small quantities of target DNA, generating larger samples for analysis. Developed in the 1980s by Kary Mullis, it revolutionized molecular biology and has since evolved through various adaptations.1 These improved the technique’s accuracy, sensitivity and specificity, expanding its use across a myriad of biological disciplines, from clinical diagnostics to forensic science.2,3 DNA amplification is achieved through repeated cycles in a thermal cycler. Each cycle involves three basic steps: denaturation, annealing and extension.4 qPCR, or real-time PCR, is a variation of conventional PCR. It tracks DNA amplification in real-time using a fluorescent tag, allowing the quantification of DNA present after each PCR cycle.2,5 One such adaptation is the development of quantitative PCR (qPCR), which enables real-time tracking of DNA amplification. This infographic introduces the key principles of qPCR, its applications, strengths and limitations. THE OF qPCR ANALYSIS HOW DOES qPCR DIFFER FROM CONVENTIONAL PCR? dNTPs Primer 5’ 5’ 3’ Template DNA New DNA strand DNA polymerase PCR qPCR Starting materials DNA template Nucleotides Primers DNA template Nucleotides Primers Fluorescent primers or probes bind to target sequences on the DNA template. Primer fluorescence is only expressed due to the presence of DNA polymerase in the extension phase.2,5 Process Analysis Subsequent analysis is required to visualize results. Gel electrophoresis separates DNA fragments based on their size and charge, allowing researchers to confirm the presence of amplified target DNA and estimate its length.6 A qPCR curve shows the quantification cycle (Cq) – a number at which fluorescence breaches the threshold level. A low Cq value indicates high amounts of the target sequence.2,5 Real-time data acquired from each qPCR thermal cycle allows researchers to track amplification as it occurs, providing precise quantification and rapid results. This characterizes the technique as highly sensitive and specific, enabling the method to have widespread Use cases and applications to various fields, from oncology, environmental, food and GMO testing, cell and gene therapy to neurobiology.7, 8, 9,10,11 APPLICATIONS OF qPCR While it has some limitations, this technique offers notable sensitivity and specificity, along with several other strengths that make it a highly valuable tool for analysis.16,17,18 qPCR is a powerful, versatile tool for analyzing DNA with high sensitivity and precision. By enabling rapid, accurate detection across many applications, including gene expression and pathogen identification, qPCR supports critical research and diagnostics, making it an essential technology in molecular biology and medicine. WHY USE qPCR? STRENGTHS AND LIMITATIONS THE POWER OF qPCR STRENGTHS LIMITATIONS • Rapid turnaround time • High sensitivity • High specificity • Quantitative results • Multiplexing suitability • Requires high-quality nucleic acids • Limited throughput • Lower standardization • Higher technical complexity • Reagent expense Copy number variation12 qPCR applications Drug target validation15 Gene expression analysis14 Biomarker detection13 Mutation detection7 Pathogen detection and quantification9 Definition Identifying specific sequence changes Definition Identifying infectious agents in samples Definition Identifying molecular disease indicators Definition Confirming the role of targets in disease Use cases Allele-specific, highly sensitive assays Use cases Rapid, precise identification of pathogens Use cases Sensitive, specific detection of targets Definition Measuring RNA levels to study gene activity Use cases Quantifies cDNA precisely in real time Definition Assessing gene gains or losses in the genome Use cases Accurate quantification of DNA copies Use cases Measures expression changes with high precision Laura Hemmingham, PhD Illustrated by Alisha Vroom Fluorescence C q Threshold 1. Initiation phase 2. Exponential phase 3. Plateau phase