Summary of the May 2013 issue of BioTechniques
The May 2013 issue of BioTechniques will feature articles describing new methods for: (i) detecting outliers in metabolite NMR spectra data, (ii) purification of chemically labeled nucleotides, (iii) calibration of antibody binding on protein microarrays, (iv) capture of coding gene sequences from divergent species for next-generation sequencing, and (v) analysis of receptor activation using a pair-wise agonist scanning flow cytometry approaches. In addition, the May issue will also feature a Tech News article examining the latest tools and applications in the growing field of metabolomics.
Solution nuclear magnetic resonance (NMR) spectroscopy is widely applied to the analysis of complex mixtures of organic compounds such as biological fluids and tissue extracts. However, targeted profiling approaches with reliable compound quantification are still hard to accomplish due to signal overlap and other interferences. In the May 2013 issue of BioTechniques, a team of researchers present a tool for automated compound quantification from pre-processed 1D and 2D heteronuclear single quantum coherence (HSQC) NMR spectral data and concomitant validation of results. The performance of this new tool is tested on a urinary spike-in dataset and compared with other quantification strategies.
In a Benchmark article slated for the May issue, a team from Seoul National University describe a facile, efficient, cost-effective and rapid purification method for chemically labeled oligonucleotide. This method eliminates the time-consuming and inefficient conventional procedures, such as ethanol precipitation and size-exclusion chromatography, of un-reacted fluorophore removal that is required to retrieve the labeled DNA once DNA-fluorophore conjugation reaction is performed. Using n-butanol saturated with water, un-reacted free fluorophores in the reaction mixture are partitioned to organic phase while the labeled DNA remains in aqueous phase. This phase extraction method is very simple and fast, and multiple samples can be handled at the same time, thereby raising the prospect of being used as a high-throughput labeling strategy.
In an effort to address the issue of quantification for antibody assays with protein microarrays, a team of researchers from Arizona State University describes the development of a Microarray Nonlinear Calibration (MiNC) method that quantifies antibody binding to the surface of microarray spots. MiNC significantly increased the linear dynamic range and reduced assay variations. In a proof of concept experiment, serological analysis of guinea pig Mycobacterium tuberculosis models showed that a larger number of candidates were identified with MiNC, which is consistent with the improved assay performance of protein microarrays.
DNA hybridization capture combined with next generation sequencing can be used to determine the DNA sequences of hundreds of target genes across hundreds of individuals in a single experiment. However, the approach has thus far only been successfully applied to sequences from closely related individuals. In the May issue, researchers from the College of Charleston and University of York detail a series of modifications that extend the reach of the method to comparisons among evolutionarily highly divergent organisms which has important implications for comparative biology.
Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. In a Report slated for May, researchers at the University of Pennsylvania describe a multiplexed Pairwise Agonist Scanning (PAS) method of measuring platelet inside-out responses to all pairs of 6 platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE2) used at their EC50 concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y1, P2Y12, TP, and IP receptors. The 3-color flow cytometry method simultaneously measured integrin αIIbβ3 activation with PAC-1 antibody, P-Selectin exposure (via alpha granule release) with anti-P-selectin, and phosphatidylserine exposure with annexinV. This method has the potential for efficiently scanning for patient-specific responses across a broad agonist-receptor space.
Keywords: next-generation sequencing, flow cytometry, metabolite analysis, metabolomics, NMR, mass spectrometry, target capture, sequence capture, antibodies, protein microarrays, linear calibration, nucleotide labeling, nucleotide purification, phase extraction