Inhibition of real-time RT–PCR quantification due to tissue-specific contaminants
Introduction
Reverse transcription–polymerase chain reaction (RT–PCR) is the method of choice for quantifying low abundant mRNAs in material such as cells and tissues [1], [2], [3], [4]. This method is fast and highly reproducible. Further, its high sensitivity is its principal advantage over other techniques.
In real-time PCR the quantification takes place within an exponential phase of the amplification curve [5]. A crossing point (CP) or threshold cycle (Ct) is then extrapolated to determine a starting amount of template molecules. The CP gives the researcher the first raw information about the expression level of a given gene.
All methods of gene quantification report their findings relative to a measurable base (e.g. copies per cell, weight of tissue, volume of blood, etc.). The correct choice of the denominator depends on the question asked and can significantly affect the quality of the results [6]. To obtain an actual number of copies, various ‘absolute’ standards are often employed [7], [8], [9], but even in these cases, the quantification is always relative as some errors in a protocol are inevitably present [6], [10]. So called housekeeping or maintenance genes [11] such as actins, tubulins, albumins, ubiquitin, glyceraldehyd-3-phosphate dehydrogenase (GAPDH), 18S or 28S ribosomal subunits (rRNA) are often used as relative standards [12]. These genes are believed to undergo little, if any, variation in expression under most experimental treatments. Yet, there have been many reports on the regulation of these genes [12], [13], [14].
Another important criterion for reliable measurement and comparison of more than one gene is that all of the genes amplify equally. Experiments using normalization with housekeeping genes often overlook this parameter despite the fact that corrections have already be suggested in the literature [15], [16], [17], [18], [19].
Many factors present in samples as well as exogenous contaminants have been shown to inhibit PCR (review in Refs. [20], [21]). For example, the presence of hemoglobin, fat, glycogen, cell constituents, Ca2+, DNA or RNA concentration, and DNA binding proteins are important factors [20], [21]. Additionally, exogenous contaminants such as glove powder and phenolic compounds from the extraction process or the plastic ware can have an inhibiting effect [20], [21].
Since some experiments compare gene expression in different organs [9], [22], tissue-specific inhibition of DNA amplification may be important. To study the amplification inhibition associated with three randomly chosen tissue types we proposed a mathematical model describing the DNA amplification kinetics in real-time PCR. Using this model we could compare parameters of the amplification kinetics and analyze them statistically.
Section snippets
Preparation of cDNA samples
Samples of cerebellum, muscle and liver were gathered from six slaughtered cows, immediately frozen in liquid nitrogen and then stored at −80 °C until the total RNA extraction procedure was performed.
Tissue samples were homogenized and total RNA was extracted with a commercially available product, peqGOLD TriFast (Peqlab, Erlangen, Germany), utilizing a single modified liquid separation procedure [23]. No additional purification was performed. Constant amounts of 1000 ng of RNA were
Results and discussion
All primers used could satisfactorily amplify the flanked sequence. The melting curve analysis and gel analysis detected very little, if any, nonspecific product. We approximated the PCR amplification kinetics with the four-parametric sigmoid model. This model describes well (in all data sets r2>0.99,n=40) the entire fluorescence curve and therefore its beginning and end do not need to be arbitrarily delimited [19]. Nevertheless, correlation between values of b and r2 showed that there were
Acknowledgements
The experimental animals were slaughtered at the EU-official slaughterhouse of the Bayerische Landesanstalt für Tierzucht at Grub, 85580 Poing, Germany.
References (32)
Real-time quantitative PCR
Methods
(2001)- et al.
Tissue-specific expression pattern of bovine prion: quantification using real-time RT–PCR
Molecular and Cellular Probes
(2003) - et al.
Housekeeping genes as internal standards: use and limits
Journal of Biotechnology
(1999) - et al.
Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT–PCR
Journal of Biochemical and Biophysical Methods
(2000) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))
Methods
(2001) - et al.
A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR
Journal of Molecular Diagnostics
(2001) - et al.
Inhibition of PCR by components of food sample, microbial diagnostic assay and DNA-extraction solutions
International Journal of Food Microbiology
(1992) - et al.
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
Analytical Biochemistry
(1997) - et al.
Developments in quantitative PCR
Clinical Chemistry and Laboratory Medicin
(1998) - et al.
A novel method for real time quantitative RT–PCR
Genome Research
(1996)
Quantitative RT–PCR: pitfalls and potential
BioTechniques
Quantification on the lightcycler instrument
Quantitative or semi-quantitative PCR: reality versus myth
PCR Methods and Applications
Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT–CR
Biotechnology Letters
Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays
Journal of Molecular Endocrinology
Quantitative RT–PCR: limits and accuracy
BioTechniques
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