Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

BACKGROUND: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of +/-25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4-8 determinations from each amplification reaction. FINDINGS: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. CONCLUSIONS: Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted

A Method for Distinguishing Between Transiently Accreting Neutron Stars and Black Holes, in Quiescence

We fit hydrogen atmosphere models to the X-ray data for four neutron stars (three from a previous paper, plus 4U 2129+47) and six black hole candidates (A0620-00, GS 2000+25, GS 1124-68, GS 2023+33, GRO J1655-40, and GRO J0422+32). While the neutron stars are similar in their intrinsic X-ray spectra (similar effective temperatures and emission area radii ~10 km), the spectra of two black hole candidates are significantly different, and the spectra of the remaining four are consistent with a very large parameter space that includes the neutron stars. The spectral differences between the neutron stars and black hole candidates favors the interpretation that the quiescent neutron star emission is predominantly thermal emission from the neutron star surface. Our work suggests that an X-ray spectral comparison in quiescence provides an additional means for distinguishing between neutron stars and black holes. The faint X-ray sources in globular clusters are also a class of objects which can be investigated in this manner.Comment: 33 pages, including 3 ps figures, LaTeX. To appear in Ap

Standard Cosmology and the BATSE Number vs. Peak Flux Distribution

The observed 2B BATSE distribution is consistent with the faintest GRBs in our sample originating from a redshift of Zmax ~ 0.8-3.0 (90\%), with the most likely values in the range of 1.0-2.2, and is largely insensitive to Omega for models with no evolution. To constrain the model parameter Omega to the range 0.1-1.0 using only Log N -- Log P distributions, more than 4000 GRBs, with a most likely value of ~ 9,000 GRBs to BATSE sensitivity. This requires a live integration time of >6 years with BATSE. Detectors sensitive to much lower limits (~ 70-400 in sensitivity) require ~ 200 GRBs, with <0.03 year 4pi ster coverage. We place limits on the amount of frequency density and, separately, peak luminosity evolution in the sample of GRBs. We find that frequency density evolution models can place the faintest GRBs at Zmax ~ 10-200, without conflicting with the observations of relative time dilation of ~ 2.Comment: 30 pages, uuencoded gzipped Postscript, 533Kb, also available from http://space.mit.edu/home/rutledge/Welcome.html ; MNRAS in pres

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

Background: Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings: Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions: The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples

Discovery of a candidate quiescent low-mass X-ray binary in the globular cluster NGC 6553

This paper reports the search for quiescent low-mass X-ray binaries (qLMXBs) in the globular cluster (GC) NGC 6553 using an XMM-Newton observation designed specifically for that purpose. We spectrally identify one candidate qLMXB in the core of the cluster, based on the consistency of the spectrum with a neutron star H-atmosphere model at the distance of NGC 6553. Specifically, the best-fit radius found using the three XMM European Photon Imaging Camera spectra is R_NS=6.3(+2.3)(-0.8) km (for M_NS=1.4 Msun) and the best-fit temperature is kTeff=136 (+21)(-34) eV. Both physical parameters are in accordance with typical values of previously identified qLMXBs in GC and in the field, i.e., R_NS~5-20 km and kTeff~50-150 eV. A power-law (PL) component with a photon index Gamma=2.1(+0.5)(-0.8) is also required for the spectral fit and contributes to ~33% of the total flux of the X-ray source. A detailed analysis supports the hypothesis that the PL component originates from nearby sources in the core, unresolved with XMM. The analysis of an archived Chandra observation provides marginal additional support to the stated hypothesis. Finally, a catalog of all the sources detected within the XMM field of view is presented here.Comment: 10 pages, 5 figures, 3 tables. Accepted to ApJ (to be published in August 2011

Chandra Observation of Quiescent Low-Mass X-ray Binaries in the Globular Cluster NGC 6304

This paper presents the analysis of candidate quiescent low mass xray binarie (qLMXBs) observed during a short Chandra/ACIS observation of the globular cluster (GC) NGC 6304. Two out of the three candidate qLMXBs of this cluster, XMMU 171433-292747 and XMMU 171421-292917, lie within the field of view. This permits comparison with the discovery observation of these sources. The one in the GC core -- XMMU 171433-292747 -- is spatially resolved into two separate X-ray sources, one of which is consistent with a pure H-atmosphere qLMXB, and the other is an X-ray power-law spectrum source. These two spectral components separately account for those observed from XMMU 171433-292747 in its discovery observation. We find that the observed flux and spectral parameters of the H-atmosphere spectral components are consistent with the previous observation, as expected from a qLMXB powered by deep crustal heating. XMMU 171421-292917 also has neutron star atmosphere spectral parameters consistent with those in the XMM-Newton observation and the observed flux has decreased by a factor 0.54^{+0.30}_{-0.24}.Comment: Accepted for publication in ApJ. 6 pages, 1 figur

Identification of a Likely Radio Counterpart of the Rapid Burster

We have identified a likely radio counterpart to the low-mass X-ray binary MXB 1730-335 (the Rapid Burster). The counterpart has shown 8.4 GHz radio on/off behavior correlated with the X-ray on/off behavior as observed by the RXTE/ASM during six VLA observations. The probability of an unrelated, randomly varying background source duplicating this behavior is 1-3% depending on the correlation time scale. The location of the radio source is RA 17h 33m 24.61s; Dec -33d 23' 19.8" (J2000), +/- 0.1". We do not detect 8.4 GHz radio emission coincident with type II (accretion-driven) X-ray bursts. The ratio of radio to X-ray emission during such bursts is constrained to be below the ratio observed during X-ray persistent emission at the 2.9-sigma level. Synchrotron bubble models of the radio emission can provide a reasonable fit to the full data set, collected over several outbursts, assuming that the radio evolution is the same from outburst to outburst, but given the physical constraints the emission is more likely to be due to ~hour-long radio flares such as have been observed from the X-ray binary GRS 1915+105.Comment: 28 pages, 4 figures; accepted for publication in ApJ (no changes

Identification of A Transient Neutron Star in Quiescence in the Globular Cluster NGC 5139

Using the Chandra/ACIS-I detector, we have identified an X-ray source (CXOU 132619.7-472910.8) in the globular cluster NGC 5139 with a thermal spectrum identical to that observed from transiently accreting neutron stars in quiescence. The absence of intensity variability on timescales as short as 4 seconds (< 25% rms variability) and as long as 5 years (<50% variability) supports the identification of this source as a neutron star, most likely maintained at a high effective temperature (approximately 1e6 K) by transient accretion from a binary companion. The ability to spectrally identify quiescent neutron stars in globular clusters (where the distance and interstellar column densities are known) opens up new opportunities for precision neutron star radius measurements.Comment: 13 pages, 4 figures, submitted to Ap

The b Distribution and the Velocity Structure of Absorption Peaks in the Lyman-Alpha Forest

A theory is developed which relates the observed b-parameter of a Lyman-alpha absorption line to the velocity-curvature of the corresponding peak in the optical depth fluctuation. Its relation to the traditional interpretation of b as the thermal broadening width is discussed. It is demonstrated that, independent of the details of the cosmological model, the differential b distribution has a high b asymptote of $dN/db \propto b^{-m}$, where $m \geq 5$, when we make the reasonable assumption that low-curvature fluctuations are statistically favored over high-curvature ones. There in general always exist absorption lines much broader than the thermal width. We then develop a linear perturbative analysis of the optical depth fluctuation, which yields a single-parameter prediction for the full b distribution: in addition to exhibiting the general high velocity tail, it explains the observed sharp low b cut-off. The dependence of the b distribution on cosmological parameters, such as $\Omega$ and the power spectrum, and reionization history as well as observation/simulation resolution is derived and discussed.Comment: 18 pages, Latex with aaspp4.sty, submitted to Ap