Comparison Of qPCR Research Articles

Analysis of Viral Promoter Usage in EBV-Infected Cell Lines: A Comparison of qPCR Following Conventional RNA Isolation and Nuclear Run-On Assay.

To interpret the results of an epigenetic analysis in gene expression studies, it is essential to characterize the activity of the relevant promoters. According to the literature, real-time PCR assay is the most widely used method for the determination of latent EBV promoter usage. Here we describe two alternative approaches to measure the activity of viral promoters in cell lines carrying latent EBV episomes. The widespread typical approach relies on total cellular RNA isolation, whereas the nuclear run-on assay described here is based on the initial isolation of nuclei, followed by in vitro transcription in the presence of biotinylated-UTP, and purification of RNA transcripts using avidin-coated magnetic beads. Finally, both methods apply reverse transcription-based real-time PCR (i.e., quantitative polymerase chain reaction, qPCR) to quantitatively measure the amount of specific transcripts. We shall describe these methods step by step and demonstrate their use for the determination of EBER1 promoter activity in EBV-positive cell lines.

Decay rates of zoonotic pathogens and viral surrogates in soils amended with biosolids and manures and comparison of qPCR and culture derived rates

AimsThe purpose of this study was to establish inactivation decay constants of foodborne pathogens and coliphage in clay and sandy soils for future "downstream" analyses such as quantitative microbial risk analysis and to compare cultivation-dependent and -independent (e.g. qPCR) methods. Methods and resultsSalmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Escherichia coli O157:H7, and Clostridium perfringens, were seeded together with MS2 and ØX174 phages, into three waste matrices (Class B biosolids, swine lagoon effluent, cattle manure), and phosphate buffered saline (PBS) as a control, and applied to two soil types (sandy loam, clay loam) using two management practices (incorporated, surface applied). S. enterica and L. monocytogenes inactivation rates were positively affected (e.g. slower rate) by solid wastes, while C. jejuni was quickly inactivated by day 7 regardless of waste type. The use of qPCR provided more conservative inactivation rates, with qPCR-based rates typically twice as slow as cultivation-based. The effect of soil type and management were less apparent as rates were variably affected. For instance, incorporation of waste negatively impacted (e.g. faster rate) inactivation of Salmonella when measured by qPCR, while the opposite was true when measured by cultivation. Inactivation rates were organism∗waste∗soil∗management dependent since the interactions of these main effects significantly affected most combinations. ConclusionsClass B biosolids and cattle manure most often slowed inactivation when measured by cultivation, but the complex interactions between variables and organism made sweeping conclusions difficult. On the contrary cultivation-independent inactivation rates were negatively affected by solid wastes. Inactivation rates developed by cultivation-dependent and -independent assays needs further scrutiny as interprerations can vary by orders of magnitude depending on the organism∗environment combination. Significance and impact of the studyThis study compares decay rate data based on waste, soil, management and assay type which can be further used in risk assessments.

The effect of iron on growth, lipid accumulation, and gene expression profile of the freshwater microalga Chlorella sorokiniana.

The effects of iron on the growth, lipid accumulation, and gene expression profiles of the limnetic Chlorella sorokiniana CCTCC M209220 under photoautotrophy were investigated. The addition of iron up to 10(-5) mol l(-l) increased final cell densities by nearly 2-fold at 2.3 × 10(7) cells/ml, growth rate by 2-fold, and the length of the exponential phase by 5 days as compared to unsupplemented controls while 10(-3) mol l(-1) iron was toxic. The lipid content increased from 12 % for unsupplemented cultures to 33 % at 10(-4) mol l(-1) iron while the highest overall lipid yield reached 179 mg l(-1). A genefishing and qPCR comparison between the C. sorokiniana at low and high iron levels indicated increases in the expression of several genes, including carbonic anhydrase involved in microalgal cell growth, as well as acc1 and choline transporter related to lipid synthesis. This study provides insights into changes in gene expression and metabolism that accompany iron supplementation to Chlorella as well as potential metabolic engineering targets for improving growth and lipid synthesis in microalgae.

Gene expression levels in archived FFPE ovarian carcinoma samples: Comparison of qPCR with tissue microarray in a 9-core pathway predictive treatment model in serous ovarian carcinoma

Objective: We have developed a statistical model based on gene expression within 9 core cancer pathways using The Cancer Genome Atlas (TCGA) microarray dataset that predicts clinical response to cytotoxic chemotherapies at the time of progression of serous ovarian carcinoma. We assayed the applicability of our microarray signatures to gene expression in quantitative polymerase chain reaction (qPCR) with patient-matched snap frozen and formalin-fixed, paraffin-embedded (FFPE) tissue samples. Successful results will enable large-scale validation from FFPE archival tissue with abundant clinical information and follow-up.

Rapid method demonstration project at four New Jersey marine beaches using real time quantitative Polymerase Chain Reaction (qPCR)

Real time quantitative Polymerase Chain Reaction (qPCR) was used at four marine bathing beaches in New Jersey as part of a demonstration project to evaluate the potential for use of qPCR as part of a routine beach monitoring program. Split sample analyses for Enterococcus spp. using membrane filtration (MF) and qPCR were performed for 11weeks during the summer of 2011 using swimming advisories based on qPCR results. Comparison of qPCR and MF results from split samples indicated that there was an 82% overall agreement rate between the two methods. Results from the qPCR tests were available by noon the same day of sample collection and swimming advisories were posted on a dedicated website. The qPCR method can be more labor intensive and requires a higher level of training to perform, however, qPCR was able to assess beach water quality in a timelier manner compared to conventional MF techniques.

DNA-Based Faecal Dietary Analysis: A Comparison of qPCR and High Throughput Sequencing Approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l −1 water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless ‘low contamination levels’ of water samples (<200 N. fowleri cells l −1) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.