Inhibition of real-time RT–PCR quantification due to tissue-specific contaminants

https://doi.org/10.1016/j.mcp.2003.09.001Get rights and content

Abstract

Real-time reverse transcription–polymerase chain reaction (RT–PCR) is currently considered the most sensitive method to study low abundance gene expression. Since comparison of gene expression levels in various tissues is often the purpose of an experiment, we studied a tissue-linked effect on nucleic acid amplification. Based on the raw data generated by a LightCycler instrument, we propose a descriptive mathematical model of PCR amplification. This model allowed us to study amplification kinetics of four common housekeeping genes in total RNA samples derived from various bovine tissues. We observed that unknown tissue-specific factors can influence amplification kinetics but this affect can be ameliorated, in part, by appropriate primer selection.

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)

  • W.M. Freeman et al.

    Quantitative RT–PCR: pitfalls and potential

    BioTechniques

    (1999)
  • R. Rasmussen

    Quantification on the lightcycler instrument

  • F. Ferré

    Quantitative or semi-quantitative PCR: reality versus myth

    PCR Methods and Applications

    (1992)
  • M.W. Pfaffl et al.

    Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT–CR

    Biotechnology Letters

    (2001)
  • S.A. Bustin

    Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

    Journal of Molecular Endocrinology

    (2000)
  • F. Souazé et al.

    Quantitative RT–PCR: limits and accuracy

    BioTechniques

    (1996)
  • Cited by (109)

    • Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system

      2016, Journal of Virological Methods
      Citation Excerpt :

      Even though 7/59 and 1/59 VI positive tissue samples tested negative by RT-qPCR and RT-iiPCR, respectively, these samples exhibited low (≤1.5 × 103 PFU/g) or very low (100 to <10 PFU/g) viral titers that could have potentially hindered efficient detection of EAV RNA by both molecular diagnostic assays. Additionally, tissue samples frequently contain PCR inhibitors that reduce the reaction efficiency and constitute a well-recognized challenge encountered with tissue material (Bustin and Nolan, 2004; Das et al., 2009; Fleige and Pfaffl, 2006; Schrader et al., 2012; Tichopad et al., 2004). The combination of low/very low viral titers along with the presence of PCR inhibitors may very well compromise the sensitivity of molecular diagnostic assays.

    • QPCR, dPCR, NGS - A journey

      2015, Biomolecular Detection and Quantification
    • Identification, cloning and heterologous expression of a new lectin like protein in tea

      2014, Journal of Applied Research on Medicinal and Aromatic Plants
    View all citing articles on Scopus
    View full text