Various external effects on PCR amplification effiency

Quantification of Insulin-like Growth Factor-1 (IGF-1) mRNA:
Development and validation of an internally standardised competitive 
Reverse Transcription-Polymerase Chain Reaction (compRT-PCR)

M. Pfaffl, H.H.D. Meyer and H. Sauerwein  (1998) 
Exp Clin Endocrinol Diabetes. 1998;106(6): 506-513.

M. Pfaffl; F. Schwarz & H. Sauerwein (1998)
Quantification of the insulin like growth factor-1 (IGF-1) mRNA: Modulation of growth intensity by 
feeding results in inter- and intra-tissue specific differences of IGF-1 mRNA expression in steers
Experimental and Clinical Endocrinology & Diabetes 106: 513-520.


To investigate the role of local IGF-1 mRNA expression in various tissues, we developed and validated a method which allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally standardiser qRT-PCR. <>A synthetic competitive template IGF-1 standard cRNA (IGF-1 cRNA) was designed, which contains the same flanking primer sequences used to amplify the wild type IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration present in tissue RNA samples, series of 250 ng total-RNA were spiked with three known quantities of the standard IGF-1 cRNA,  incubated for competitive RT-PCR reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240 bp from the wild type IGF-1 mRNA) were subsequently separated and quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R = competitor PCR product / wild type PCR product) was plotted against the number of starting molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA present in the sample can then be read off where R = 1. The validated assay had a detection limit of 1600 IGF-1 cRNA molecules/reaction, the intra-assay variation was 7.4% (n = 5) and linearity (r = 0.997) was given between 140 ng to 840 ng total-RNA input. The present method was first applied to study the effect of long term castration on the IGF-1 expression rates in bovine tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the plasma concentrations as quantified by RIA and were higher in intact than in castrated animals. In two skeletal muscles (m. splenius and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower than in liver, respectively, without any differences between steers and bulls. In bulls, the IGF-1 mRNA expression was higher in m. splenius (p<0.01) than m. gastrocnemius, indicating that locally produced IGF-1 might be important for sexually dimorphic muscle growth patterns.
Zurück zum Seitenanfang
IGF-1 mediates the anabolic growth hormone actions in skeletal tissues. Above that locally expressed IGF-1 is an important growth regulator acting in auto- and paracrine way (Thissen et al., 1994). To investigate the tissue specific expression in low abundant tissues a method is required which allows for a reliable quantification of IGF-1 mRNA. Considering these limitations, RT-PCR offers the most potent instrument to detect low-abundance mRNAs and the detection limit can been increased up to 1000-fold in comparison to other methods, e.g. Northern hybridisation (Saiki et al., 1988). The relationship between the initial amount A of target mRNA present in the tissues and the amount Yn of DNA produced after n PCR cycles can be expressed as
Yn = A *(1+E)n
where E is the amplification efficiency of one reaction step (Chelly et al., 1988). Small variations in the reaction efficiency, therefore, translate into large differences in the amount of RT-PCR product generated after n cycles. These limitations in quantitative analyses can be compensated by parallel co-amplification of the native mRNA together with known amounts of an internal standard cRNA. The amplification efficiency should affect both templates similarly. Several designs have been used in quantitative  RT-PCR to obtain an internal standard cRNA that suits the characteristics of having an  identical amplification efficiency as the wild-type mRNA template and of being easy distinguishable from it (Nedelman et al., 1992). Hereby the construction of an internal standard by inserting (Martini et al., 1995) or deleting (Becker-Andrè and Hahlbrock, 1989; Piatak et al., 1993; Malucelli et al., 1996) a relatively small sequence within the wild type template are common practice. Due to the negative relationship between the efficiency of 
amplification and the length of the amplified sequences, the both templates should be as short as possible (Rolfs et al., 1992). Analysis and quantification of competitive PCR  products can be done either by electrophoretic separation with densitometric quantification or by HPLC and following UV detection at 260 nm. HPLC-UV is the most exact quantification method for PCR products in terms of accuracy, precision and linearity (Katz et al., 1990). In consequence we designed, developed and validated an internally standardised IGF-1 mRNA RT-PCR assay with subsequent HPLC-UV quantification for quantitative comparisons in tissues of low IGF-1 mRNA abundance. The method was first applied to investigate the effect of castration on IGF-1 mRNA expression in bovine liver and two different skeletal muscles.


Establishment and Validation of the quantitative IGF-1 mRNA  RT-PCR

Assay conditions

Considering the described criteria we designed a short internal standard IGF-1 cRNA, for which the same flanking primers are used as for the wild-type IGF-1 mRNA. The conditions for the RT-PCR as described in Materials and Methods were optimised with regard toPCR buffer pH, primer and  MgCl2 concentration in the PCR reaction, dNTPs concentration and annealing temperatures. To ensure a parallel start in all individual reaction tubes and  to increase specificity, yield and precision of the PCR, a "hot-start" amplification with a melting wax barrier between RT reagents and PCR master-mix was applied. The quantification of wild-type IGF-1 mRNA in different tissues required a preliminary estimation of the IGF-1 cRNA start-molecule concentration range  to be used for individual tissues. This was performed by 7 titration steps from 1.6 * 108 to 1.6 * 1011 cRNA start-molecules versus 250 ng total tissue RNA. For routine comparisons, three standard concentrations covering the range in which equal amounts of the two amplification products are to be expected for a certain tissue were selected.

Zurück zum Seitenanfang

Amplification efficiencies

The amplification efficiencies for the wild-type and the standard template were recorded during the exponential and the plateau phase of the PCR. Figure 3a shows the results of the competitive co-amplification for the two amplificates. Until cycle numbers 23-25 there was an exponential increase in the amount of both products, followed by the plateau phase. In order to compare the amplification efficiencies of target IGF-1 mRNA and standard IGF-1 cRNA, the 10log of the HPLC integrals (10log Yn) was plotted versus the number of  PCR cycles (abscissa) and the  linear regressions were then calculated for the  exponential and the plateau phase (Figure 3b). The relationship Yn = A * (1+E)n, in which  E is the amplification efficiency of interest, can be transformed to 10log Yn = n *  10log (1+E) + 10log A,  yielding a linear equation: y = x * a + t. The resulting efficiencies of competitive IGF-1 RT-PCR during the exponential phase were nearly identical (Figure 3b; E (IGF-1 cRNA) = 0.66 ; r = 0.98 and E (IGF-1 mRNA) = 0.65 ; r = 0.98). Similarly, during the plateau phase the amplification efficiencies were parallel with E (IGF-1 cRNA) = 0.05 (r = 0.78) and E (IGF-1 mRNA) = 0.04 (r = 0.75). The initial ratio R of the both products remained constant throughout the amplification cycles.


The sensitivity of the RT-PCR was evaluated using different starting amounts of IGF-1 cRNA  from 2.8 ag (16 IGF-1 cRNA molecules) to 28 ng (1.6 * 1011 cRNA molecules). The minimal detectable amount of IGF-1 cRNA using the HPLC-UV detection modus was 1600  molecules/tube. 

Linearity and variability
The precision of the HPLC-UV quantification of PCR products was initially established by quantifying 28 individual DNA samples at 7 different concentrations from 5 to 325 ng DNA. A linear relationship between the DNA concentration injected (d) onto the DEAE column and the respective peak integral (i) could be demonstrated (i = 1.13 * d  +  3.62; r = 0.99). The linearity of the RT-PCR was determined by quantifying the IGF-1 mRNA in serial dilutions of a liver RNA preparation (140, 280, 560 and 840 ng). Each RNA dilution was assayed together with four different IGF-1 cRNA standard concentrations. Figure 4 shows the resulting ratio plots for the four individual RNA input concentrations. The IGF-1 mRNA molecule numbers initially present were read off at R = 1. In Figure 5 the amount of IGF-1 mRNA molecules (a) measured in the different RNA dilutions is plotted versus the total-RNA input (t) into the RT-PCR assay. A linear relationship between the amount of analyte and the measured IGF-1 mRNA concentration could thus be demonstrated (a = 2.0 * 107 * t  +  5.4 * 106; r = 0.997). To confirm the reproducibility of the competitive IGF-1 RT-PCR, the assay variation was determined: five identical RT-PCR experiments were set up;  each with three different standard dilutions and 250 ng liver RNA. Quantification resulted in 1.069 ± 0.079 * 109 IGF-1 mRNA molecules (n = 5) and thus in an assay variation of 7.4%. 

Quantification of androgen receptor mRNA in tissues by competitive co-amplification
of a template
in reverse transcription-polymerase chain reaction.

Malucelli A, Sauerwein H, Pfaffl MW, Meyer HHD
J Steroid Biochem Mol Biol. 1996 Aug;58(5-6): 563-568.

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.

Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method.
Sikorsky JA, Primerano DA, Fenger TW, Denvir J.
Biochem Biophys Res Commun. 2004 Oct 22;323(3): 823-8930
Department of Microbiology, Immunology and Molecular Genetics, Joan C. Edwards
School of Medicine, Marshall University, Huntington, WV 25704, USA

Polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression. To test the effect of specific lesions on PCR amplification efficiency, we amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template. We found that while the amplification efficiencies of templates containing a single 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were not significantly perturbed, the presence of a single 8-oxo-7,8-dihydro-2'-deoxyadenosine, abasic site, or a cis-syn thymidine dimer dramatically reduced amplification efficiency. In addition, while templates containing two 8-oxodGs separated by 13 bases amplified as well as the unmodified template, the presence of two tandem 8-oxodGs substantially hindered amplification. From these findings, we conclude that the reduction in polymerase progression is dependent on the type of damage and the relative position of lesions within the template.

Amplification effiency of thermostable DNA polymerases
Bahram Arezi, Weimei Xing, Joseph A. Sorge, and Holly H. Hogrefe*
Stratagene Cloning Systems, 11011 North Torrey Pines Road, La Jolla, CA 92037, USA
Analytical Biochemistry 321 (2003) 226–235

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.

Evaluation of Real-Time PCR Amplification Efficiencies to Detect PCR Inhibitors

Elias J. Kontanis and Floyd A. Reed.

J Forensic Sci, July 2006, Vol. 51, No. 4
Real-time PCR analysis is a sensitive template DNA quantitation strategy that has recently gained considerable attention in the forensic community. However, the utility of real-time PCR methods extends beyond quantitation and allows for simultaneous evaluation of template DNA extraction quality. This study presents a computational method that allows analysts to identify problematic samples with statistical reliability by comparing the amplification efficiencies of unknown template DNA samples with clean standards. In this study, assays with varying concentrations of tannic acid are used to evaluate and adjust sample-specific amplification efficiency calculation methods in order to optimize their inhibitor detection capabilities. Kinetic outlier detection and prediction boundaries are calculated to identify amplification efficiency outliers. Sample-specific amplification efficiencies calculated over a four-cycle interval starting at the threshold cycle can be used to detect reliably the presence of 0.4 ng of tannic acid in a 25 mL PCR reaction. This approach provides analysts with a precise measure of inhibition severity when template samples are compromised. Early detection of problematic samples allows analysts the opportunity to consider inhibitor mitigation strategies prior to genotype or DNA sequence analysis, thereby facilitating sample processing in high-throughput forensic operations.

Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR.

E.J. Walsh · C. King · R. Grimes · A. Gonzalez
Biomed Microdevices (2006) 8: 59–64

The two-phase segmented flow approach to the processing and quantitative analysis of biological samples in microdevices offers significant advantages over the singlephase continuous flow methodology. Despite this, little is known about the compatibility of samples and reactants with segmenting fluids, although a number of investigators have reported reduced yield and inhibition of enzymatic reactions depending on the segmenting fluid employed. The current study addresses the compatibility of various segmenting fluids with real time quantitativePCRto understand the physicochemical requirements of this important reaction in biotechnology. The results demonstrate that creating a static segmenting fluid/PCR mix interface has a negligible impact on the reaction efficiency, crossing threshold and end fluorescence levels using a variety of segmenting fluids. The implication is then that the previously reported inhibitory effects are the result of the dynamic motion between the segmenting fluid and the sample in continuously flowing systems. The results presented here are a first step towards understanding the limitations of the segmented flow methodology, which are necessary to bring this approach into mainstream

Quantitative real-time polymerase chain reaction: methodical analysis and mathematical model.

Wilkening S, Bader A.
J Biomol Tech. 2004 Jun;15(2):107-11.
German Research Centre for Biotechnology, Braunschweig, Germany.

Real-time polymerase chain reaction was established for 16 genes using the LightCycler system to evaluate gene expression in human hepatocytes. During the experiments a large set of data has been obtained. These data have now been evaluated with respect to template stability, accuracy of melting curve analysis, and reproducibility. In addition, the statistical evaluation of the efficiencies of all 16 polymerase chain reactions led to a new mathematical model. To examine template stability, the degradation of mRNA and cDNA was determined at different temperatures. Surprisingly, cDNA, which was obtained by first-strand synthesis, appeared to degrade significantly faster than the respective mRNA. Melting curve analysis is a fast and sensitive method to check for polymerase chain reaction specificity. However, we show that two transcription variants of the glutathione S-transferase 1 gene, with over 100 bp length difference, could not be distinguished by this method. Furthermore, an equation was set up describing the correlation between polymerase chain reaction efficiency and crossing point. This equation can be used to estimate the number of template molecules without having a standard of known concentration. Finally, experimental reproducibility of the real-time polymerase chain reaction was defined.
Enhancement in the efficiency of polymerase chain reaction by TiO2 nanoparticles:
crucial role of enhanced thermal conductivity.

Abdul Khaliq R, Sonawane PJ, Sasi BK, Sahu BS, Pradeep T, Das SK, Mahapatra NR.
Nanotechnology. 2010 Jun 25;21(25):255704. Epub 2010 Jun 2.
Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India.
Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by nanoparticles is an emerging area of research. We observed that TiO(2) nanoparticles of approximately 25 nm diameter caused significant enhancement of PCR efficiency for various types of templates (namely plasmid DNA, genomic DNA and complementary DNA). By a series of experiments, the optimal TiO(2) concentration was determined to be 0.4 nM, which resulted in up to a seven-fold increase in the amount of PCR product. As much as 50% reduction in overall reaction time (by reduction of the number of cycles and the time periods of cycles) was also achieved by utilizing TiO(2) nanoparticles without compromising the PCR yield. Investigations of the mechanism of such PCR enhancement by simulations using the 'Fluent K epsilon turbulent model' provided evidence of faster heat transfer in the presence of TiO(2) nanoparticles. Consistent with these findings, TiO(2) nanoparticles were observed to augment the denaturation of genomic DNA, indicating more efficient thermal conductivity through the reaction buffer. TiO(2) nanoparticle-assisted PCR may be useful for profound reduction of the overall PCR reaction period and for enhanced amplification of DNA amplicons from a variety of samples, including GC-rich templates that are often observed to yield unsatisfactory results.