Raw Fluorescence Data Research Articles

Accurate and Efficient Data Processing for Quantitative Real-Time PCR using a Tripartite Plant Virus as a Model

Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucumber mosaic virus (CMV) genomic RNAs using raw fluorescence data of real-time PCR, and equations were proposed to compare their expression levels in virions or in planta. The results, as confirmed by standard curve and Northern blotting methods, show that the expression levels of different genes can be compared more accurately and more efficiently by these equations, especially using theoretical fluorescence (F0) and calibration factors (CF), determined by linear regression PCR (LinRegPCR). Thus, these equations, combined with data analysis by the LinRegPCR method, can greatly enhance the high-throughput quantification ability of real-time PCR, and permit accurate, reliable, and facile investigation of the changes in CMV RNAs accumulation.

Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms

Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories.

Microarray Analysis in the Archaeon Halobacterium salinarum Strain R1

BackgroundPhototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea.Methodology/Principal FindingsWe developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis.Conclusion/SignificanceThis is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

Genomics Analysis of Freshly-Purified Human Marrow Stromal Cells (MSCs): Comparison with Hematopoietic Lineage Cells and Culture-Expanded MSCs.

Gene and antigen expression profile of bone marrow MSCs in culture has been investigated, but these characteristics of MSCs in vivo are poorly understood. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (f-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (e-MSCs). Following enrichment with Anti-Fibroblast MicroBeads, f-MSCs were purified by cell sorting as CD45lowCD235a-LNGFR+ [1] and HLCs as CD45+CD235a+LNGFR-. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct comparison with qPCR. Duplicate hybridizations were used to define 3 relative zones of sensitivity and select appropriate thresholds for significant changes in expression. The microarray data were validated by qPCR using several mesenchymal lineage genes (including Sox9 and Runx2) and by flow cytometry for several known MSC surface markers (including CD73, CD90, CD146, CD10, CD63 and CD130). Expression data were further searched for gene ontology using a custom-made, Perl-based program. Compared to HLCs, f-MSCs over-expressed genes encoding extracellular matrix proteins (including versican, fibronectin and collagen I), stromal cytokines/chemokines (including SDF-1, IL-7 and CXCL10), and molecules associated with cell junctions and communication with endothelium (including connexin 43, VCAM-1 and angiopoietin-1). Osteogenesis-related genes (BMPs and their receptors, alkaline phosphatase, osteonectin, osteocalcin, osteomodulin and others) were more abundant in f-MSCs than in HLCs. Some mature fat tissue products were also quite abundant in f-MSCs (particularly lipoprotein lipase and adipsin), suggesting the presence of cells committed to adipogenesis. Of note, the expression of stroma- and fat-related transcripts was significantly reduced in e-MSCs compared to f-MSCs. Novel surface antigens of f-MSCs identified by microarray, Junctional Adhesion Molecule 2 (normally expressed on vascular endothelial cells), Leptin receptor (implicated in fat and bone metabolism), and CD81 (tetraspanin family adhesion molecule) were confirmed at the protein level by 6-colour flow cytometry. Altogether, this work confirmed the CD45lowCD235a-LNGFR+ phenotype f-MSCs in human bone marrow, revealed a unique expression profile of f-MSCs in vivo and described new surface markers of f-MSCs. These data have implications for our understanding of the in vivo biology of non-hematopoietic bone marrow stromal cells and their role in hematopoiesis and skeletal remodelling in health and disease.

Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR

BackgroundConventional real-time PCR to quantify the allele ratio in pooled DNA mainly depends on PCR amplification efficiency determination and Ct value, which is defined as the PCR cycle number at which the fluorescence emission exceeds the fixed threshold. Because of the nature of exponential calculation, slight errors are multiplied and the variations of the results seem too large. We have developed a new PCR data point analysis strategy for allele ratio quantification based on normalized fluorescence ratio.ResultsIn our method, initial reaction background fluorescence was determined based upon fitting of raw fluorescence data to four-parametric sigmoid function. After that, each fluorescence data point was first subtracted by respective background fluorescence and then each subtracted fluorescence data point was divided by the specific background fluorescence to get normalized fluorescence. By relating the normalized fluorescence ratio to the premixed known allele ratio of two alleles in standard samples, standard linear regression equation was generated, from which unknown specimens allele ratios were extrapolated using the measured normalized fluorescence ratio. In this article, we have compared the results of the proposed method with those of baseline subtracted fluorescence ratio method and conventional Ct method.ConclusionResults demonstrated that the proposed method could improve the reliability, precision, and repeatability for quantifying allele ratios. At the same time, it has the potential of fully automatic allelic ratio quantification.

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction

Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of RNA transcript abundance. Useful data from this method depend on fitting data to theoretical curves that allow computation of mRNA levels. Calculating accurate mRNA levels requires important parameters such as reaction efficiency and the fractional cycle number at threshold (CT) to be used; however, many algorithms currently in use estimate these important parameters. Here we describe an objective method for quantifying qRT-PCR results using calculations based on the kinetics of individual PCR reactions without the need of the standard curve, independent of any assumptions or subjective judgments which allow direct calculation of efficiency and CT. We use a four-parameter logistic model to fit the raw fluorescence data as a function of PCR cycles to identify the exponential phase of the reaction. Next, we use a three-parameter simple exponent model to fit the exponential phase using an iterative nonlinear regression algorithm. Within the exponential portion of the curve, our technique automatically identifies candidate regression values using the P-value of regression and then uses a weighted average to compute a final efficiency for quantification. For CT determination, we chose the first positive second derivative maximum from the logistic model. This algorithm provides an objective and noise-resistant method for quantification of qRT-PCR results that is independent of the specific equipment used to perform PCR reactions.

SAS Programs for Real-Time RT-PCR Having Multiple Independent Samples

Relative real-time reverse transcription PCR (RT-PCR) has become an important tool for quantifying changes in messenger RNA (mRNA) populations following differential development or stimulation of tissues or cells. However, the best methods for conducting such experiments and analyzing the resultant data remain an issue of discussion. In this report we describe an appropriate experimental methodology and the computer programs necessary to generate a meaningful statistical analysis of the combined biological and experimental variability in such experiments. Specifically, logarithmic transformations of raw fluorescence data from the log-linear portion of real-time PCR growth curves for both target and reference genes are analyzed using a SAS/STAT Mixed Procedure program specifically designed to give a point estimate of the relative expression ratio of the target gene with associated 95% confidence interval. The program code is open-source and is printed in the text.

Combined Phase Singularity and Wavefront Analysis for Optical Maps of Ventricular Fibrillation

Much of the research into the mechanisms of ventricular fibrillation (VF) employs high-resolution mapping of electrical activation and recovery patterns. We previously developed a method for analyzing electrically mapped VF patterns that was based on identifying individual VF wavefronts. We now introduce a related method designed to take into account the information on repolarization that is present in optically mapped VF data. The new method first converts raw fluorescence data to an angular variable that tracks the phase of the mapped tissue through the depolarization-repolarization cycle. We define wavefronts in this context as isolines of phase that terminate either at boundaries or at singular points within the phase field. These singularities are the pivots of functional reentry and are important determinants of VF patterns. We parameterize VF by constructing data structures that describe wavefronts and singularities and also maintain wavefront-wavefront, wavefront-singularity, and singularity-singularity relationships. We describe one important application of this parameterization, which is to identify, localize, and characterize the importance of occurrences of propagation block during VF.

Effects of incident light intensity on the yield of steady-state chlorophyll fluorescence in intact leaves. An example of bioenergetic homeostasis

The emission of modulated 685-nm chlorophyll fluorescence from intact leaves was measured using a pulsed (yellow) exciting light of very low intensity combined with various non-modulated lights of sub- and supersaturating intensities. It was found that, whereas the “extreme” (initial and maximal) fluorescence levels were drastically quenched by high light intensities, the level of steady-state modulated fluorescence in light-adapted leaves was virtually constant and independent of the intensity and the quality of the continuous light used to drive photosynthesis. These results provided an example of biological homeostasis. For instance, the modulated fluorescence emission from a dark-adapted leaf with all photosystem II reaction centers in the open configuration was similar to the steady-state modulated fluorescence emission measured in leaves illuminated with an additional strong, photosynthetically saturating light with all photosystem II traps closed. This homeostatic behavior was characteristic of only healthy and mature leaves. It was strongly perturbed in young leaves and also in leaves exposed to unfavorable environmental conditions. In some stress situations (e.g. leaf dehydration), noticeable deviations from homeostasis were detected before any significant changes in the familiar photochemical ( q p) and non-photochemical ( q NP) fluorescence quenching coefficients could be observed, suggesting that the analysis of this “bio-energetic homeostasis”, revealed by the examination of raw fluorescence data, could be a useful approach for early detection of subtle changes in the physiological state of plants.