title: Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications.
abstract: There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies.
authors: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors: Orsolya
authors: O
authors: Kolacsek
authors: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors: Enikő
authors: E
authors: Pergel
authors: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors: Nóra
authors: N
authors: Varga
authors: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors: Ágota
authors: Á
authors: Apáti
authors: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. Electronic address: orban.tamas@ttk.mta.hu.
authors: Tamás I
authors: TI
authors: Orbán
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics*; D018628:Gene Dosage; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D055647:Rats, Transgenic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data
chemical_list: D004251:DNA Transposable Elements; D012333:RNA, Messenger
keywords: Copy number; PGBD3; Promoter activity; Quantitative PCR; Sleeping Beauty; Splice variant