Real-time PCR offers a wide area of application to analyze the role of gene
activity in various biological aspects at the molecular level with higher specificity,
sensitivity and the potential to troubleshoot with post-PCR processing and difficulties.
With the recent advancement in the development of functional tissue graft for the
regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour
and differentiation over tissue construct toward specific lineage through analyzing the
expression of an array of specific genes. With the ability to collect data in the
exponential phase, the application of Real-Time PCR has been expanded into various
fields such as tissue engineering ranging from absolute quantification of gene
expression to determine neo-tissue regeneration and its maturation. In addition to its
usage as a research tool, numerous advancements in molecular diagnostics have been
achieved, including microbial quantification, determination of gene dose and cancer
research. Also, in order to consistently quantify mRNA levels, Northern blotting and in
situ hybridization (ISH) methods are less preferred due to low sensitivity, poor
precision in detecting gene expression at a low level. An amplification step is thus
frequently required to quantify mRNA amounts from engineered tissues of limited size.
When analyzing tissue-engineered constructs or studying biomaterials–cells
interactions, it is pertinent to quantify the performance of such constructs in terms of
extracellular matrix formation while in vitro and in vivo examination, provide clues
regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time
PCR; recent Protocol used in the lab, primer designing, detection methods and
troubleshooting of the experimental problems.
Keywords: Biomaterial, Coding DNA sequences (CDS), DNA, FASTA,
Fluorescence, Hybridization, Molecular diagnostics, Master mix, RNA, Real
Time-PCR, SYBER green, Tissue Engineering, Thermostability, TaqMan, Wound
healing.
