High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.     

Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs

Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.     

Fragment size difference between multiplex and singleplex PCR products and their practical implications

By simultaneously amplifying several loci in the same reaction, multiplex PCR has been used in gene mapping and DNA typing with polymorphic short tandem repeat loci. Previous studies have discussed in detail the various parameters and conditions that influence the quantity of individual products generated by multiplex PCR. In practice, when a primer pair fails to amplify in a multiplex PCR for some individuals, singleplex PCR is often employed as a supplement to amplify the primer pair. However, the reliability of this procedure is unknown. In this study, we used six primer pairs from ABI PRISM Linkage Mapping Set version 2 to perform multiplex and singleplex reactions. The fluorescence-labeled amplification products were separated and detected on ABI PRISM 310 Genetic Analyzer. We found that for the marker D1S468, multiplex and singleplex reactions for the majority of individuals yielded reactions of different sizes. Therefore, the potential size difference between multiplex and singleplex reactions needs to be investigated. This investigation is essential to employ multiplex PCR supplemented with singleplex PCR in gene mapping and DNA typing.     

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader® mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1β. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z′ factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader® assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.     

A sensitive multiplex assay for piRNA expression

PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10 pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II

J.‐H. Lee, N.‐W. Lee, S.‐W. Hong, Y.‐S. Nam, J.‐W. Choi and Y.‐S. Kim Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II Objective: To establish an efficient multiplex real‐time PCR assay for 15 human papillomavirus (HPV) genotypes, we designed multiplexing parameters and compared our PCR system with the hybrid capture (HC) II test using cervical cytology samples. Methods: For preventing cross‐reactive amplifications, variable HPV genes (E1, E2, E6, E7 and L1) were targeted. The melting temperatures of all primers and probes, and the size of the PCR product were optimized for the multiplex PCR. Our PCR system was compared with the HC II assays in the detection and genotyping of HPV infection using 173 cytology smears. Discordant cases between the two assays were verified by direct HPV DNA sequencing. Results: Of 173 women, 93 (53.8%) were HPV‐positive by the HC II assay and/or the multiplex real‐time PCR assay. The HPV genotypes were determined in 92 (98.9%) of 93 cases by the multiplex real‐time PCR and/or DNA sequencing. The agreement rate between multiplex PCR and HC II methods was 91.9% (kappa = 0.84). Although the sample size of this study needs to be increased to have epidemiological significance, multiple infections and HPV 16 were the predominant type. HPV 58, 52 and 18 accounted for 25% of HPV infections. HPV 52, 58 and 31 constituted 30% of CIN 2/3. Conclusion: The multiplex real‐time PCR system shows a good and reliable clinical performance. This in house PCR assay is fast and cost‐effective for HPV genotyping and the detection of HPV co‐infection in the post‐HPV vaccination era.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Multi-node graphs: a framework for multiplexed biological assays.

Multiplex polymerase chain reaction (PCR) is an extension of the standard PCR protocol in which primers for multiple DNA loci are pooled together within a single reaction tube, enabling simultaneous sequence amplification, thus reducing costs and saving time. Potential cost saving and throughput improvements directly depend on the level of multiplexing achieved. Designing reliable and highly multiplexed assays is challenging because primers that are pooled together in a single reaction tube may cross-hybridize, though this can be addressed either by modifying the choice of primers for one or more amplicons, or by altering the way in which DNA loci are partitioned into separate reaction tubes. In this paper, we introduce a new graph formalism called a multi-node graph, and describe its application to the analysis of multiplex PCR scalability. We show, using random multi-node graphs that the scalability of multiplex PCR is constrained by a phase transition, suggesting fundamental limits on efforts to improve the cost-effectiveness and throughput of standard multiplex PCR assays. In particular, we show that when the multiplexing level of the reaction tubes is roughly theta(log (sn)) (where s is the number of primer pair candidates per locus and n is the number of loci to be amplified), then with very high probability we can 'cover' all loci with a valid assignment to one of the tubes in the assay. However, when the multiplexing level of the tube exceeds these bounds, there is no possible cover and moreover the size of the cover drops dramatically. Simulations using a simple greedy algorithm on real DNA data also confirm the presence of this phase transition. Our theoretical results suggest, however, that the resulting phase transition is a fundamental characteristic of the problem, implying intrinsic limits on the development of future assay design algorithms.     

Real-time PCR: duplexing without optimization

Real-time PCR applications generally require determination of the threshold cycle of a gene of interest in parallel with an endogenous control. In standard situations, the gene-specific and control reactions are run as separate samples (singleplex). In contrast, duplex approaches combine both reactions within a single well, thereby saving time, cost, and material. However, establishing duplex reactions usually requires laborious optimization justifiable only for the analysis of large sample series. Hence, in research settings, singleplex approaches are used most commonly. To establish conditions for duplexing without the need of optimization, we tested the performance of 40 premade TaqMan gene expression assays in duplex reactions with an endogenous control using three different polymerase mixes. The results were compared with singleplex reactions. Duplexing results obtained with one of the multiplex polymerase mixes correlated extremely well (r(2)=0.95) with the singleplex reference. The findings of our study demonstrate that the combination of this polymerase mix and premade gene expression assays will yield reliable and reproducible results in duplex approaches without preceding optimization.     

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus,Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.