Abstract
In this chapter we cover what we have found to be a "best practice" for real-time polymerase chain reaction (PCR) for relative mRNA quantification. We describe our techniques for tissue-sample collection and freezing, sample handling for quick and reproducible extraction of total RNA, first strand cDNA synthesis, real-time PCR amplification, and template dilution and storage for PCR. We offer our insights on intron-spanning primer design for genes (when applicable), effective primer selection vs reaction optimization, and relative quantification and sample normalization using housekeeping genes. Comments are also provided on the choice of PCR reagents including fluorescent probes, prevention of PCR "carry over," and on the practical aspects of real-time PCR theory and interpretation.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
