Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Statistical methods for efficiency adjusted real-time PCR quantification

The statistical treatment for hypothesis testing using real-time PCR data is a challenge for quantification of gene expression. One has to consider two key factors in precise statistical analysis of real-time PCR data: a well-defined statistical model and the integration of amplification efficiency (AE) into the model. Previous publications in real-time PCR data analysis often fall short in integrating the AE into the model. Novel, user-friendly, and universal AE-integrated statistical methods were developed for real-time PCR data analysis with four goals. First, we addressed the definition of AE, introduced the concept of efficiency-adjusted Delta Delta Ct, and developed a general mathematical method for its calculation. Second, we developed several linear combination approaches for the estimation of efficiency adjusted Delta Delta Ct and statistical significance for hypothesis testing based on different mathematical formulae and experimental designs. Statistical methods were also adopted to estimate the AE and its equivalence among the samples. A weighted Delta Delta Ct method was introduced to analyze the data with multiple internal controls. Third, we implemented the linear models with SAS programs and analyzed a set of data for each model. In order to allow other researchers to use and compare different approaches, SAS programs are included in the Supporting Information. Fourth, the results from analysis of different statistical models were compared and discussed. Our results underline the differences between the efficiency adjusted Delta Delta Ct methods and previously published methods, thereby better identifying and controlling the source of errors introduced by real-time PCR data analysis.     

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.     

Quantitative biomarker analysis of synovial gene expression by real-time PCR

Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1β and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents.     

Evaluation of procedures for amplification of small-size samples for hybridization on microarrays

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.     

Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.     

标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量.     

Preservation of Gene Expression Ratios Among Multiple Complex cDNAs After PCR Amplification: Application to Differential Gene Expression Studies

Comparative gene expression studies are often limited by low availability of tissue and poor quality of extractable mRNA. Collective PCR amplification of minute quantities of mRNA has great potential for overcoming these limitations. However, there remains significant concern about the effects of amplification on the absolute and relative abundance of individual mRNAs that could complicate subsequent gene expression studies. To address this problem, we systematically compared the relative abundance of many specific mRNAs from complex cDNA preparations (from tissue and cultured cells) both before and after amplification by PCR. Our results demonstrated that, as expected, the absolute abundance of different mRNAs in a cDNA library is altered in an unpredictable manner by PCR amplification. However, we found that the concentration ratios of specific mRNAs among different cDNA preparations were routinely well conserved after PCR amplification. Thus, for the purpose of comparative expression studies for specific mRNAs in two (or more) complex cDNAs, PCR-amplified cDNA is equally useful as unamplified cDNA. These results provide a rigorous experimental validation and offer a theoretical treatment to support the utility of PCR amplified cDNA for differential gene expression studies. We conclude that the inherent difficulties in performing differential screening studies such as gene chip and array analyses on limited amounts of biological materials can be overcome by a PCR amplification step without compromising data quality. This revised version was published online in July 2006 with corrections to the Cover Date.     

Shape based kinetic outlier detection in real-time PCR

Background  

Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

Transgene copy number comparison in recombinant mammalian cell lines: critical reflection of quantitative real-time PCR evaluation

Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.     

A standard curve based method for relative real time PCR data processing

Background  

Currently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR.     

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The F?rster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.     

A direct PCR approach to accelerate analyses of human-associated microbial communities

Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.     

Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data

Amplification of a cDNA product by quantitative polymerase chain reaction (qPCR) gives rise to fluorescence sigmoidal curves from which absolute or relative target gene content of the sample is inferred. Besides comparative C(t) methods that require the construction of a reference standard curve, other methods that focus on the analysis of the sole amplification curve have been proposed more recently. Among them, the so-called sigmoidal curve fitting (SCF) method rests on the fitting of an empirical sigmoidal model to the experimental amplification data points, leading to the prediction of the amplification efficiency and to the calculation of the initial copy number in the sample. The implicit assumption of this method is that the sigmoidal model may describe an amplification curve quantitatively even in the portion of the curve where the fluorescence signal is hidden in the noise band. The theoretical basis of the SCF method was revisited here for defining the class of experimental amplification curves for which the method might be relevant. Applying the SCF method to six well-characterized different PCR assays illustrated possible pitfalls leading to biased estimates of the amplification efficiency and, thus, of the target gene content of a sample.     

A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

Background

Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach.

Results

Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison with the LinReg and Miner automated qPCR data processing packages further demonstrated the superior performance of this kinetic-based methodology.

Conclusion

Called "linear regression of efficiency" or LRE, this novel kinetic approach confers the ability to conduct high-capacity absolute quantification with unprecedented quality control capabilities. The computational simplicity and recursive nature of LRE quantification also makes it amenable to software implementation, as demonstrated by a prototypic Java program that automates data analysis. This in turn introduces the prospect of conducting absolute quantification with little additional effort beyond that required for the preparation of the amplification reactions.     

小麦脱水素基因wzy2表达量实时荧光定量PCR方法的建立和应用

以陕合6号小麦品种为材料,用SYBR GreenⅡ染料,参照小麦脱水素wzy2和-βactin基因序列设计引物,建立小麦wzy2基因的实时荧光定量PCR分析方法.结果表明:通过对标准品标准曲线和熔解曲线分析,其标准曲线的Ct值检测范围为12~30,PCR扩增效率高达111.3%;不同的标准品扩增曲线的间距为3.078,接近于理想值3.32,且可显示产物特异的单峰,引物的特异性扩增强.小麦脱水素基因wzy2表达量实时荧光定量PCR的测定结果表明,小麦脱水素基因wzy2在胁迫24 h的表达量明显高于胁迫12 h的表达量.