Marcus Gassmann

Bio: Marcus Gassmann is an academic researcher from Agilent Technologies. The author has contributed to research in topics: Gel electrophoresis of nucleic acids & Glycomics. The author has an hindex of 9, co-authored 18 publications receiving 2469 citations. Previous affiliations of Marcus Gassmann include Stony Brook University & Florida International University.

The RIN: an RNA integrity number for assigning integrity values to RNA measurements

Abstract: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.

High-Throughput Profiling of the Serum N-Glycome on Capillary Electrophoresis Microfluidics Systems: Toward Clinical Implementation of GlycoHepatoTest

Abstract: We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use.

Microfluidic assembly with coupled microfluidic devices

Abstract: A microfluidic assembly (1;57) has at least one microfluidic flow path (17,27,29,35,38;82,85,95) and at least one inlet port (11,13,15;81,97,101) coupled to the flow path (17,27,29,35,38;82,85,95). The microfluidic assembly (1;57) has a first microfluidic device (7;63) that executes a microfluidic process and has a second microfluidic device (9;61). The microfluidic assembly (1;57) has an interface (5;65). The interface (5;65) couples the first microfluidic device (7;63) and the second microfluidic device (9;61).

The development of mini pentameric STR loci for rapid analysis of forensic DNA samples on a microfluidic system

Abstract: There is increasing interest in developing methods for portable DNA analysis in mass disasters and criminal identification. Currently most forensic STR DNA analysis is performed by CE; however, these instruments are not portable and require long sample run times. One potential solution is the development of microfluidic systems for DNA typing. Unfortunately, fairly long (ca. 20 cm) separation channels are usually required for the proper resolution of multiplexed STR loci used in human identification. Commercially available systems like the Agilent 2100 Bioanalyzer have a small footprint and utilize chips with shorter channels and reduced resolution. Such portable systems might be valuable for evidence screening in remote locations. However, due to their lower resolution, most standard 4 base STR loci and their inherent 2 base variants will not resolve on such systems. In this paper, we discuss the development of reduced length pentameric (5 base) STR amplicons. Pentameric STRs have fewer variant alleles and are easier to separate due to the wider spacing between alleles. By incorporating novel denaturing sieving polymers in a short microfluidic channel, we demonstrate efficient separations on these chips. Such an approach can serve as a useful tool for rapid microfluidic DNA typing.

Reversible thermo-responsive sieving matrix for oligonucleotide separation

Abstract: A reversible thermo-responsive gel system, consisting of Pluronic copolymer mixture of F87 and F127, has been used to successfully carry out the separation of oligonucleotides, for the first time, by microchip-based capillary electrophoresis. Pluronic triblock copolymers F87 (E(61)P(40)E(61)) and F127 (E(99)P(69)E(99)), with E, P, and subscript denoting oxyethylene, oxypropylene, and segment length respectively, have a unique temperature dependent viscosity-adjustable property and a dynamic coating ability in aqueous solution, including 1 x TBE buffer. The mixture solution has a reversible thermo-responsive property and its sol-gel transition temperature can be adjusted ranging from about 17 degrees C to 38 degrees C by varying the relative weight ratio of F87 and F127 at an optimized concentration of approximately 30% (w/v) for oligonucleotide separations. Oligonucleotide sizing markers ranging from 8 to 32 base could be successfully separated in a 1.5 cm long separation channel by the mixture solution in its gel-like state. A 30% (w/v) with a F87/F127 weight ratio of 1 ratio 2 which has a "sol-gel" transition point of about 26 degrees C shows the best sieving ability. The sieving ability of the mixture solution was further confirmed in an Agilent Bioanalyzer 2100 system. Fast separation of oligonucleotides has been achieved within 40 s with one base resolution.

Quantification of mRNA using real-time RT-PCR

Abstract: The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase.

Abstract: Neurodegenerative disorders such as Parkinson and Alzheimer disease cause motor and cognitive dysfunction and belong to a heterogeneous group of common and disabling disorders. Although the complex molecular pathophysiology of neurodegeneration is largely unknown, major advances have been achieved by elucidating the genetic defects underlying mendelian forms of these diseases. This has led to the discovery of common pathophysiological pathways such as enhanced oxidative stress, protein misfolding and aggregation and dysfunction of the ubiquitin-proteasome system. Here, we describe loss-of-function mutations in a previously uncharacterized, predominantly neuronal P-type ATPase gene, ATP13A2, underlying an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia (PARK9, Kufor-Rakeb syndrome). Whereas the wild-type protein was located in the lysosome of transiently transfected cells, the unstable truncated mutants were retained in the endoplasmic reticulum and degraded by the proteasome. Our findings link a class of proteins with unknown function and substrate specificity to the protein networks implicated in neurodegeneration and parkinsonism.

Review: Activation patterns of microglia and their identification in the human brain

Abstract: Microglia in the central nervous system are usually maintained in a quiescent state. When activated, they can perform many diverse functions which may be either beneficial or harmful depending on the situation. Although microglial activation may be accompanied by changes in morphology, morphological changes cannot accurately predict the function being undertaken by a microglial cell. Studies of peripheral macrophages and in vitro and animal studies of microglia have resulted in the definition of specific activation states: M1 (classical activation) and M2 (sometimes subdivided into alternative activation and acquired deactivation). Some authors have suggested that these might be an overlapping continuum of functions rather than discrete categories. In this review, we consider translational aspects of our knowledge of microglia: specifically, we discuss the question as to what extent different activation states of microglia exist in the human central nervous system, which tools can be used to identify them and emerging evidence for such changes in ageing and in Alzheimer's disease.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues

Abstract: Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22ha, which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project

Abstract: The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community.