Evaluation of the LightCycler® 1536 Instrument for high-throughput quantitative real-time PCR

Quantitative real-time PCR (qPCR) is a frequently used, sensitive and accurate method to study gene expression profiles. However, its throughput was so far limited for routine laboratories to 384 reactions per run based on the limitations of the available instruments. Recently, the LightCycler® 1536 Instrument was launched providing a high-throughput solution for qPCR with the analysis of 1536 reactions in approximately 45 min. We assessed the accuracy and sensitivity of this novel technology for the analysis of gene expression profiles in combination with the Innovadyne? Nanodrop? Express pipetting robot. We compared expression profiles obtained for 42 genes in 71 samples between the Universal ProbeLibrary and the LightCycler® 1536 Instrument and SYBR Green I and the ABI PRISM 7900HT system. We found that the results were highly reproducible between both systems. Beside the higher throughput, the advantage of the LightCycler® 1536 Instrument was the reduced consumption of reagents and sample material.     

Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.     

Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases

The provision of prenatal diagnosis requires the highest standards in laboratory practice to ensure an accurate result. In preimplantation genetic diagnosis protocols additionally have to address the need to achieve an accurate result from 1 to 2 cells within a limited time. Emerging protocols of "non-invasive" prenatal diagnosis, which are based on analysis of free fetal DNA in the circulation of the pregnant mother, also have to achieve a result from a limited quantity of fetal DNA against a high background of maternal free DNA. Real-time PCR uses fluorescent probes or dyes and dedicated instruments to monitor the accumulation of amplicons produced throughout the progress of a PCR reaction. Real-time PCR can be used for quantitative or qualitative evaluation of PCR products and is ideally suited for analysis of nucleotide sequence variations (point mutations) and gene dosage changes (locus deletions or insertions/duplications) that cause human monogenic diseases. Real-time PCR offers a means for more rapid and potentially higher throughput assays, without compromising accuracy and has several advantages over end-point PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detection with a high degree of sensitivity. This review will focus on real-time PCR protocols that are suitable for genotyping monogenic diseases with particular emphasis on applications to prenatal diagnosis, non-invasive prenatal diagnosis and preimplantation genetic diagnosis.     

Genotyping of single nucleotide polymorphisms in cytokine genes using real-time PCR allelic discrimination technology

Single nucleotide polymorphisms (SNPs), particularly those within regulatory regions of genes that code for cytokines often impact expression levels and can be disease modifiers. Investigating associations between cytokine genotype and disease outcome provides valuable insight into disease etiology and potential therapeutic intervention. Traditionally, genotyping for cytokine SNPs has been conducted using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), a low throughput technique not amenable for use in large-scale cytokine SNP association studies. Recently, Taqman real-time PCR chemistry has been adapted for use in allelic discrimination assays. The present study validated the accuracy and utility of real-time PCR technology for a number of commonly studied cytokine polymorphisms known to influence chronic inflammatory diseases. We show that this technique is amenable to high-throughput genotyping and overcomes many of the problematic features associated with PCR-RFLP including post-PCR manipulation, non-standardized assay conditions, manual allelic identification and false allelic identification due to incomplete enzyme digestion. The real-time PCR assays are highly accurate with an error rate in the present study of <1% and concordance rate with PCR-RFLP genotyping of 99.4%. The public databases of cytokine polymorphisms and validated genotyping assays highlighted in the present study will greatly benefit this important field of research.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

High throughput gene expression measurement with real time PCR in a microfluidic dynamic array

We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays.     

Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages

Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad’s CFX96 real-time system and Applied Biosystems’ Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad’s CFX96 real-time system and Applied Biosystems’ Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.     

Real-time PCR quantification of precursor and mature microRNA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

焦测序技术的研究进展

焦测序技术是一种实时DNA测序技术。它在DNA聚合酶、三磷酸腺苷硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶4种酶的协同作用下,将焦磷酸转化为等量的荧光信号,通过荧光信号的高低实时检测待测序列,操作简便,可实现高通量、自动化测定,检测不需要电泳,不需要对样品标记和染色,结果准确可靠重复性好。本文综述了焦测序技术的基本原理、历史及其在测序模板制备技术、反应体系和检测仪器三个方面的最新进展,并重点介绍制备单链的Late-PCR技术、高灵敏度反应体系的获得以及454公司超高通量测序技术,并对焦测序技术的发展做了展望。     

Assessment of the specificity of Burkholderia and Pseudomonas qPCR assays for detection of these genera in soil using 454 pyrosequencing

In this study, two highly specific quantitative PCR assays targeting the bacterial genera Burkholderia and Pseudomonas were developed and evaluated on soil samples. The primers were targeting different multivariate regions of the 16S rRNA gene and designed to be compatible with quantitative PCR and the high throughput 454 pyrosequencing technique. The developed assays were validated using the standard methods. All tests with the new developed assays showed very high specificity. Pyrosequencing was used for direct analysis of the PCR product and applied as a specificity measurement of the primers. The Pseudomonas primers showed a 99% primer specificity, which covered 200 different Pseudomonas sequence clusters in 0.5 g of soil. In contrast to that the same approach using the genus-specific Burkholderia primers showed only 8% primer specificity. This discrepancy in primer specificity between the normal procedures compared with pyrosequencing illustrates that the common validation procedures for quantitative PCR primers may be misleading. Our results exemplify the fact that current 16S RNA gene sequence databases might lack resolution within many taxonomic groups and emphasize the necessity for a standardized and functional primer validation protocol. A possible solution to this could be to supplement the normal verification of quantitative PCR assays with a pyrosequencing approach.     

High-resolution semi-quantitative real-time PCR without the use of a standard curve

The repeatability and sensitivity of a simple, adaptable, semi-quantitative, real-time RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the LightCycler real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.     

A comparative analysis of PCR-based detection methods for avaian malaria

Here, 4 polymerase chain reaction (PCR) assays are compared to test for the presence of avian malaria, including both the Plasmodium and Haemoproteus genera, in 29 different species of African rainforest birds. Two of these PCR assays use primer sets that amplify fragments of the cytochrome b (cyt b) gene of Plasmodium; the other 2 target the 18S ribosomal subunit gene. These PCR assays were performed using genomic DNA extracted from blood and subsequently compared with the results obtained by microscopic examination of blood smears taken from the same individuals. The 2 primer sets amplifying the cyt b gene were found to perform more reliably than those that target the 18S rRNA gene and yielded a substantial number of positive samples that were undetected by blood smear analysis. Of all the individuals screened by PCR, 40% tested positive for avian malaria, whereas 27% tested positive by blood smear analysis. Although sequence variation in the parasites may prohibit the specific alignment of primers and the subsequent PCR amplification of some individuals, PCR, once optimized, is faster, cheaper, and more reliable than blood smear analysis for large-scale screening.     

Comparative metabolomic analysis of <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp. C4 during the degradation of phenanthrene

We describe a quantitative analysis of the genetic diversity of phenol-degrading potential in bacterial communities from laboratory-scale activated sludge. Genomic DNA extracted from activated sludge from two sequential batch reactors fed with synthetic sewage plus phenol was amplified using conserved primers for the major subunit of the phenol hydroxylase (LmPH) gene and used to generate clone libraries. Following phylogenetic analysis, 59 sequences containing a 470-bp fragment clustered into six distinct subgroups with a genetic distance of 8%, most likely representing ecologically relevant variants of the enzyme. Seven sets of primers were designed to target the six clusters and used to obtain quantitative information on the dynamics of LmPH gene diversity using real-time PCR assays throughout 9 months of bioreactors operation. Total LmPH gene copy number remained approximately steady in phenol-amended and control reactors. However, a significant increase in phenol-degrading activity in the phenol-amended sludge was accompanied by a parallel increase in LmPH gene diversity, suggesting that phenol degradation in the activated sludge depends on the combined activity of a number of redundant species.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.