%0 Journal Article
%T Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong?
%A Rasmus Skern
%A Petter Frost
%A Frank Nilsen
%J BMC Molecular Biology
%D 2005
%I BioMed Central
%R 10.1186/1471-2199-6-10
%X Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions.The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.Reverse transcription (RT) followed by quantitative polymerase chain reaction (Q-PCR) is at present the most sensitive method for transcript abundance measurement. However, there are many sources of errors, both when purifying RNA, performing the RT reaction and during the PCR setup [3,4]. Q-PCR utilises optical measurement of generated amplicons to survey PCR amplifications. It is common to derive the initial template concentration from the number of amplification cycles required for a signal to reach a threshold chosen by the investigator [1,2,5]. In relative quantification the expression of a target gene is stated relative to a standard gene, which is assumed to be constitutively and uniformly expressed. One popular approach, the 2-¦¤¦¤CT method, assumes ¡Ö100% efficient target and standard gene PCR reactions given that the results conform to certain criteria [1,5]. In recognition of the fact that PCR efficiencies may vary between runs or between target and standard genes, other numerous methods have emerged that calculate template concentrations using amplification simulations or PCR efficiencies derived from CT values or fluorescence data [2,6-9]. We here present the results of a case study showing that the interpretation of results may vary dramatic
%U http://www.biomedcentral.com/1471-2199/6/10