title: Real-time reverse-transcription polymerase chain reaction: technical considerations for gene expression analysis.
abstract: The reverse transcription - polymerase chain reaction (RT-PCR) is a sensitive technique for the quantification of steady-state mRNA levels, particularly in samples with limited quantities of extracted RNA, or for analysis of low level transcripts. The procedure amplifies defined mRNA transcripts by taking advantage of retroviral enzymes with reverse transcriptase (RT) activity, coupled to PCR. The resultant PCR product concentration is directly proportional to the initial starting quantity of mRNA, therefore allowing quantification of gene expression by incorporation of a fluorescence detector for the appropriate amplicons. In this chapter, we describe a number of the most popular techniques for performing RT-PCR and detail the subsequent analysis methodologies required to interpret the resultant data in either a relative manner or through absolute quantification of gene expression levels.
authors.affiliation: DNA Damage Research Group, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales, UK. s.h.doak@swansea.ac.uk
authors.forename: Shareen H
authors.initials: SH
authors.lastname: Doak
authors.forename: Zoulikha M
authors.initials: ZM
authors.lastname: Zaïr
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003959:Diamines; D005456:Fluorescent Dyes / Q000032:analysis; D015870:Gene Expression; D006801:Humans; D009930:Organic Chemicals / Q000032:analysis; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I