Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab microL(-1) milk and 0.4 ng mL(-1) Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n = 8) or non-transgenic (n = 7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

A communication and information technology infrastructure for real time monitoring and management of type 1 diabetes patients

This paper is focused on the integration of state-of-the-art technologies in the fields of telecommunications, simulation algorithms, and data mining in order to develop a Type 1 diabetes patient's semi to fully-automated monitoring and management system. The main components of the system are a glucose measurement device, an insulin delivery system (insulin injection or insulin pumps), a mobile phone for the GPRS network, and a PDA or laptop for the Internet. In the medical environment, appropriate infrastructure for storage, analysis and visualizing of patients' data has been implemented to facilitate treatment design by health care experts.

A real time simulation model of glucose-insulin metabolism for type 1 diabetes patients

In this paper, a simulation model of glucose-insulin metabolism for Type 1 diabetes patients is presented. The proposed system is based on the combination of Compartmental Models (CMs) and artificial Neural Networks (NNs). This model aims at the development of an accurate system, in order to assist Type 1 diabetes patients to handle their blood glucose profile and recognize dangerous metabolic states. Data from a Type 1 diabetes patient, stored in a database, have been used as input to the hybrid system. The data contain information about measured blood glucose levels, insulin intake, and description of food intake, along with the corresponding time. The data are passed to three separate CMs, which produce estimations about (i) the effect of Short Acting (SA) insulin intake on blood insulin concentration, (ii) the effect of Intermediate Acting (IA) insulin intake on blood insulin concentration, and (iii) the effect of carbohydrate intake on blood glucose absorption from the gut. The outputs of the three CMs are passed to a Recurrent NN (RNN) in order to predict subsequent blood glucose levels. The RNN is trained with the Real Time Recurrent Learning (RTRL) algorithm. The resulted blood glucose predictions are promising for the use of the proposed model for blood glucose level estimation for Type 1 diabetes patients.

A relation between screening masses and real-time rates

Thermal screening masses related to the conserved vector current are determined for the case that the current carries a non-zero Matsubara frequency, both in a weak-coupling approach and through lattice QCD. We point out that such screening masses are sensitive to the same infrared physics as light-cone real-time rates. In particular, on the perturbative side, the inhomogeneous Schrödinger equation determining screening correlators is shown to have the same general form as the equation implementing LPM resummation for the soft-dilepton and photon production rates from a hot QCD plasma. The static potential appearing in the equation is identical to that whose soft part has been determined up to NLO and on the lattice in the context of jet quenching. Numerical results based on this potential suggest that screening masses overshoot the free results (multiples of 2πT) more strongly than at zero Matsubara frequency. Four-dimensional lattice simulations in two-flavour QCD at temperatures of 250 and 340 MeV confirm the non-static screening masses at the 10% level. Overall our results lend support to studies of jet quenching based on the same potential at T ≳ 250 MeV.

Real-time simulation of large open quantum spin systems driven by dissipation

We consider a large quantum system with spins 12 whose dynamics is driven entirely by measurements of the total spin of spin pairs. This gives rise to a dissipative coupling to the environment. When one averages over the measurement results, the corresponding real-time path integral does not suffer from a sign problem. Using an efficient cluster algorithm, we study the real-time evolution from an initial antiferromagnetic state of the two-dimensional Heisenberg model, which is driven to a disordered phase, not by a Hamiltonian, but by sporadic measurements or by continuous Lindblad evolution.

Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR.

BACKGROUND Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. RESULTS We describe a qPCR technique based on the single copy gene β' DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. CONCLUSIONS This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

Time-Resolved Ultra–High Resolution Optical Coherence Tomography for Real-Time Monitoring of Selective Retina Therapy

Purpose: Selective retina therapy (SRT) is a novel treatment for retinal pathologies, solely targeting the retinal pigment epithelium (RPE). During SRT, the detection of an immediate tissue reaction is challenging as tissue effects remain limited to intracellular RPE photodisruption. Time-resolved ultra-high axial resolution optical coherence tomography (OCT) is thus evaluated for the monitoring of dynamic optical changes at and around the RPE during SRT. Methods: An experimental OCT system with an ultra-high axial resolution of 1.78 µm was combined with an SRT system and time-resolved OCT M-scans of the target area were recorded from four patients undergoing SRT. OCT scans were analyzed and OCT morphology was correlated with findings in fluorescein angiography, fundus photography and cross-sectional OCT. Results: In cases where the irradiation caused RPE damage proven by fluorescein angiography, the lesions were well discernible in time-resolved OCT images but remained invisible in fundus photography and cross-sectional OCT acquired after treatment. If RPE damage was introduced, all applied SRT pulses led to detectable signal changes in the time-resolved OCT images. The extent of optical signal variation seen in the OCT data appeared to scale with the applied SRT pulse energy. Conclusion: The first clinical results proved that successful SRT irradiation induces detectable changes in the OCT M-scan signal while it remains invisible in conventional ophthalmoscopic imaging. Thus, real-time high-resolution OCT is a promising modality to monitor and analyze tissue effects introduced by selective retina therapy and may be used to guide SRT in an automatic feedback mode.

Real-time simulation of nonequilibrium transport of magnetization in large open quantum spin systems driven by dissipation

Using quantum Monte Carlo, we study the nonequilibrium transport of magnetization in large open strongly correlated quantum spin-12 systems driven by purely dissipative processes that conserve the uniform or staggered magnetization, disregarding unitary Hamiltonian dynamics. We prepare both a low-temperature Heisenberg ferromagnet and an antiferromagnet in two parts of the system that are initially isolated from each other. We then bring the two subsystems in contact and study their real-time dissipative dynamics for different geometries. The flow of the uniform or staggered magnetization from one part of the system to the other is described by a diffusion equation that can be derived analytically.

Real-time computer-based visual feedback improves visual acuity in downbeat nystagmus - a pilot study.

BACKGROUND Patients with downbeat nystagmus syndrome suffer from oscillopsia, which leads to an unstable visual perception and therefore impaired visual acuity. The aim of this study was to use real-time computer-based visual feedback to compensate for the destabilizing slow phase eye movements. METHODS The patients were sitting in front of a computer screen with the head fixed on a chin rest. The eye movements were recorded by an eye tracking system (EyeSeeCam®). We tested the visual acuity with a fixed Landolt C (static) and during real-time feedback driven condition (dynamic) in gaze straight ahead and (20°) sideward gaze. In the dynamic condition, the Landolt C moved according to the slow phase eye velocity of the downbeat nystagmus. The Shapiro-Wilk test was used to test for normal distribution and one-way ANOVA for comparison. RESULTS Ten patients with downbeat nystagmus were included in the study. Median age was 76 years and the median duration of symptoms was 6.3 years (SD +/- 3.1y). The mean slow phase velocity was moderate during gaze straight ahead (1.44°/s, SD +/- 1.18°/s) and increased significantly in sideward gaze (mean left 3.36°/s; right 3.58°/s). In gaze straight ahead, we found no difference between the static and feedback driven condition. In sideward gaze, visual acuity improved in five out of ten subjects during the feedback-driven condition (p = 0.043). CONCLUSIONS This study provides proof of concept that non-invasive real-time computer-based visual feedback compensates for the SPV in DBN. Therefore, real-time visual feedback may be a promising aid for patients suffering from oscillopsia and impaired text reading on screen. Recent technological advances in the area of virtual reality displays might soon render this approach feasible in fully mobile settings.

Real-Time In Vivo Characterization of Primary Liver Tumors With Diffuse Optical Spectroscopy During Percutaneous Needle Interventions: Feasibility Study in Woodchucks

OBJECTIVE This study presents the first in vivo real-time optical tissue characterization during image-guided percutaneous intervention using near-infrared diffuse optical spectroscopy sensing at the tip of a needle. The goal of this study was to indicate transition boundaries from healthy tissue to tumors, namely, hepatic carcinoma, based on the real-time feedback derived from the optical measurements. MATERIALS AND METHODS Five woodchucks with hepatic carcinoma were used for this study. The woodchucks were imaged with contrast-enhanced cone beam computed tomography with a flat panel detector C-arm system to visualize the carcinoma in the liver. In each animal, 3 insertions were performed, starting from the skin surface toward the hepatic carcinoma under image guidance. In 2 woodchucks, each end point of the insertion was confirmed with pathologic examination of a biopsy sample. While advancing the needle in the animals under image guidance such as fluoroscopy overlaid with cone beam computed tomography slice and ultrasound, optical spectra were acquired at the distal end of the needles. Optical tissue characterization was determined by translating the acquired optical spectra into clinical parameters such as blood, water, lipid, and bile fractions; tissue oxygenation levels; and scattering amplitude related to tissue density. The Kruskal-Wallis test was used to study the difference in the derived clinical parameters from the measurements performed within the healthy tissue and the hepatic carcinoma. Kurtoses were calculated to assess the dispersion of these parameters within the healthy and carcinoma tissues. RESULTS Blood and lipid volume fractions as well as tissue oxygenation and reduced scattering amplitude showed to be significantly different between the healthy part of the liver and the hepatic carcinoma (P < 0.05) being higher in normal liver tissue. A decrease in blood and lipid volume fractions and tissue oxygenation as well as an increase in scattering amplitude were observed when the tip of the needle crossed the margin from the healthy liver tissue to the carcinoma. The kurtosis for each derived clinical parameter was high in the hepatic tumor as compared with that in the healthy liver indicating intracarcinoma variability. CONCLUSIONS Tissue blood content, oxygenation level, lipid content, and tissue density all showed significant differences when the needle tip was guided from the healthy tissue to the carcinoma and can therefore be used to identify tissue boundaries during percutaneous image-guided interventions.

Strategic Behavior in Day-Ahead and Real-Time Markets for Electricity: Offer Cost or Payment Cost Minimization?

This study compares the procurement cost-minimizing and productive efficiency performance of the auction mechanism used by independent system operators (ISOs) in wholesale electricity auction markets in the U.S. with that of a proposed alternative. The current practice allocates energy contracts as if the auction featured a discriminatory final payment method when, in fact, the markets are uniform price auctions. The proposed alternative explicitly accounts for the market clearing price during the allocation phase. We find that the proposed alternative largely outperforms the current practice on the basis of procurement costs in the context of simple auction markets featuring both day-ahead and real-time auctions and that the procurement cost advantage of the alternative is complete when we simulate the effects of increased competition. We also find that a trade-off between the objectives of procurement cost minimization and productive efficiency emerges in our simple auction markets and persists in the face of increased competition.