High-throughput Droplet PCR

Philip Day, Amelia Markey, Stephan Mohr
University of Manchester, United Kingdom

Abstract
The polymerase chain reaction has facilitated the ready analysis of nucleic acids. The depth of sequence analysis that can be reached via PCR is largely independent of sensitivity requirements, and more a function of the analyte intricacy within the biological sample. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. Avoidance of bulk measurement has been made possible through adopting principles of digital PCR where samples are diluted to achieve a homogeneous target, which is often presented as a single molecular target. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The generation of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to form discrete packages for the purpose of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. The packaging into droplets also enables the measurement of nucleic acids from defined populations of cells from bio-samples, or molecules of nucleic acids from the individual nuclei of cells, by means of high throughput, single cell analysis. Details are provided for the production of microfluidic devices for both creating droplets and undertaking PCR in a continuously flowing microfluidic stream. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration to achieve the conditions favouring efficient PCR.


Back to High Throughput quantitative PCR – digital PCR

Rapid Quantification of DNA libraries for Next Generation Sequencing

Raza Ahmed
Agilent Technologies, United Kingdom

Abstract
Next-generation DNA sequencing workflows require an accurate quantification of the DNA molecules to be sequenced for optimal performance of the instrument. Here, we demonstrate the use of qPCR for quantification of DNA libraries used in next-generation sequencing. We find that the quantification data generated with the Bioanalyzer and qPCR are comparable for high quality library preparations. We also show that qPCR quantification may allow improvements to current NGS workflows, including reducing the amount of library DNA required, increasing the accuracy in quantifying amplifiable DNA, and avoiding amplification bias by reducing or eliminating the need to amplify DNA before sequencing.


Back to High Throughput quantitative PCR – digital PCR

Prognostic Impact of Gene Expression Analyses in Human Glioblastoma- MGMT expression analysis

Simone Kreth
LMU Munich, Germany

Abstract
O6-Methylguanine-DNA-methyltransferase (MGMT) is a unique protein, which repairs DNA damage caused by alkylating agents. It has recently been linked to the therapeutic success of alkylating agent chemotherapy, such as temozolomide treatment: The relative expression of MGMT in the tumor may determine response to therapy. Epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression. In human malignant gliomas – tumors with an extremely poor prognosis and rapid progression rates- it was shown that MGMT promoter methylation is correlated with improved progression-free and overall survival after temozolomide treatment. Thus, MGMT promoter methylation status is used as predictive factor for survival benefit of glioma patients from temozolomide therapy, based on the assumption that epigenetic silencing of the MGMT promoter leads to a decreased MGMT protein expression with a diminished ability of the tumour to repair chemotherapy-induced lesions. The real clinical impact of this assumption still is controversial as in 20 – 30 % of all cases MGMT CGI methylation status does not correlate with the clinical response to chemotherapy. In particular the rather high number of patients with response to treatment despite a non-methylated MGMT promoter indicates that the MGMT methylation status cannot yet be used to exclude patients from chemotherapy. In the current prospective study, we tested the predictive impact of MGMT mRNA expression in malignant glioma (65 patients) after radiotherapy and/or chemotherapy with temozolomide and its correlation with MGMT promoter methylation status. As a prerequisite, we first evaluated adequate reference genes that are stably expressed in malignant glioma using the RealTime ready Human Reference Gene Panel (Roche). MGMT mRNA expression was discordant to MGMT promoter methylation status in 30% of the evaluated tumors. In particular, 15% of the unmethylated tumors expressed unexpected low levels of MGMT mRNA; Low MGMT mRNA expression turned out to be an independent and strong prognostic factor for time to progression and length of survival in univariate and multivariale models (p<0.0001); patients with an unmethyleted tumor and low MGMT mRNA expression did significantly better than those with high mRNA expression. The determination of MGMT mRNA expression is a powerful method for predictive evaluation of malignant glioma patients undergoing chemotherapy with temozolomide. In particular, it allows to further stratify those patients with an unmethylated MGMT promoter.

Back to High Throughput quantitative PCR – digital PCR

Western-type diet affects the expression of genes known to be involved in human colorectal carcinogenesis in histologically normal mucosa of ApcMin/+ mice

Marjaana Pussila1, Sarantaus Laura1, Päivärinta Essi2, Ollila Saara1, Dermadi Bebek Denis1, Niku Mikael2, Nyström Minna1
1 University of Helsinki, Department of Biological and Environmental Sciences, Genetics, Helsinki, Finland; 2 University of Helsinki, Department of Food and Environmental Sciences, Nutrition, Helsinki, Finland

Abstract
Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and its incidence is increasing in all industrialized countries. The development of CRC is a multistep process. Over the past recent years, evidence has accumulated indicating that apart from genetic alterations, epigenetic alterations play a major role in the initiation and progression of CRC, also in the hereditary colon cancer syndromes. Epigenetics refers to heritable changes in phenotype or gene expression without a change in the genetic code itself, as opposed to genetics, which refers to information transmitted in gene sequences. The best-known epigenetic marker is DNA methylation, and diet is a major aspect of the environment that may influence DNA methylation thus providing an important link between cancer and nutrition. Based on human and experimental evidence it has been concluded that Western-type diet (WD) may predispose gut mucosa to colon cancer. Here, we evaluated the effects of WD on the early phases of intestinal carcinogenesis by using the ApcMin/+ (multiple intestinal neoplasia) mouse model, which is analogous to human familial adenomatous polyposis (FAP) (syndrome). ApcMin/+ mice were assigned randomly into two dietary groups [WD (n=12) or AIN-93G diet (n=14)] at the age of 5 weeks. WD is a modified AIN-93G that contains more fat, especially saturated fat, and less calcium, fiber, vitamin D and folic acid. Mice were sacrificed at the age of 15 weeks, and the small and large intestines were removed and opened. Samples from histologically normal mucosa were collected both from distal small and large intestine. Using custom StellARray qPCR arrays (Lonza), the expression of 94 genes previously demonstrated to play a role in human colorectal carcinogenesis, and many of which have been reported to be aberrantly methylated during the carcinogenic process, was analyzed and compared between the two diet groups (WD vs. AIN-93G). The expression of 5 genes out of the 94 studied was found to be significantly different between the two diet groups in both parts of the intestine (fold change 1.3 – 2.9; p<0.033). The expression of one gene linked to fatty acid metabolism was increased in the WD group as compared to control diet group both in the small and the large intestine. Otherwise, the two parts of intestine showed different spectra of altered gene expression. In summary, based on observed mRNA expression differences, 9 candidate genes for CRC initation were detected by using StellARray qPCR arrays. Further studies, e.g. methylation analyses, will be performed to survey the biological significance of the findings.

Back to High Throughput quantitative PCR – digital PCR

Detection of Related AKT Family Members using Solaris qPCR Gene Expression Assays

Ian Kavanagh1, Zaklina Strezoska2, Melissa Kelley2
1 Thermo Fisher Scientific, Epsom, Surrey, UK; 2 Thermo Fisher Scientific, Lafayette, CO, USA

Abstract
qPCR is now performed in every segment of life science research and utilizing high performance assays is essential for generating high confidence data. Solaris™ qPCR Gene Expression Assays are ideal for routine molecular applications because they combine minor groove binder (MGB) and Superbase technologies with a rigorous design algorithm for detection of all known splice variants of a gene target, while distinguishing among closely related family members. Here we describe an application of this new probe-based detection technology, in a study of the highly related AKT gene family members in regulating the activation and redistribution of the Forkhead/FOXO1 transcription factor.


Back to High Throughput quantitative PCR – digital PCR

Digital PCR and intra-cellular expression profiling

Mikael Kubista 1 2, Monika Sidova 1,2, David Svec 1,2, Radek Sindelka 1,2
1 TATAA Biocenter; 2 Institute of Biotechnology, Czech Academy of Sciences

Abstract
Expression profiling of single cells reveal substantial heterogeneity of unrelated mRNAs, while transcripts with related functions show correlated expression levels, evidencing stringent temporal control of genes’ expressions. In my talk I address the spatial distribution of mRNAs within a cell. The Xenopus laevis oocyte is excellent model for these studies, because of its large size, high content of mRNA, and differentially colored poles, which allow the cell to be aligned, mounted, and sliced. Measuring the mRNA levels in the slices with RT qPCR reveals three classes of mRNAs with distinct special distributions. The presence of these intra cellular mRNA gradients is further confirmed with digital PCR. Our finding suggests that before cell division mRNAs may become spatially distributed within the cells such that the progeny cells obtain different mRNA contents and thus become predisposed for differentiation. We postulate this is general mechanism behind asymmetric cell division.


Download a free GenEx trial version on GenEx.gene-quantification.info



Back to High Throughput quantitative PCR – digital PCR