Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.

Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.

Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

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The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we com- pared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively glob- al protein expression in cells and tissues.

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Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

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Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

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Global analysis of protein expression in yeast

To gauge the current commitment to scientific research in the United States of America (US), we compared federal research funding (FRF) with the US gross domestic product (GDP) and industry research spending during the past six decades. In order to address the recent globalization of scientific research, we also focused on four key indicators of research activities: research and development (RD and b) the ability to produce beneficial innovations for society: “How will the US continue to foster its strengths?”

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The United States of America and Scientific Research

Data-intensive science will open up new avenues to explore, new questions to ask, and new ways to answer. Yet, this potential cannot be unlocked without new emphasis on education of the researchers gathering data, the analysts analyzing data and the cross-disciplinary participants working together to make it happen. This article is a summary of the education issues and challenges of data-intensive sciences and cloud computing as discussed in the Data-Intensive Science (DIS) workshop in Seattle, September 19–20, 2010.

Education and data-intensive science in the beginning of the 21st century.

One perspective on the emergence of the child prodigy is derived from comparing the role of unique forms of abstraction in those disciplines in which the child has been wondrously creative. Music, mathematics, poetry, and computer programming have all witnessed the child as a creative force; biology has not. Perhaps it is the lack of a suitable abstraction, symbols, and rules for their use that hampers both the child prodigy and the expert in their pursuit of understanding living cells. Repurposing existing languages has yet to accelerate that understanding; perhaps it is time to nudge the evolution of language to better serve the description and prediction of the behavior of living systems.

https://stke.sciencemag.org/content/sigtrans/2004/222/pe9.full.pdf

From Play to Laws: Language in Biology

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B. Robert Franza

Papers

The United States of America and Scientific Research

Education and data-intensive science in the beginning of the 21st century.

From Play to Laws: Language in Biology

p210 bcr-abl In Vivo Signalling by Oncoprotein Tyrosine Kinase Protein Tyrosine Phosphatase 1B Antagonizes