60 research outputs found
Specific and non specific hybridization of oligonucleotide probes on microarrays
Gene expression analysis by means of microarrays is based on the sequence
specific binding of mRNA to DNA oligonucleotide probes and its measurement
using fluorescent labels. The binding of RNA fragments involving other
sequences than the intended target is problematic because it adds a "chemical
background" to the signal, which is not related to the expression degree of the
target gene. The paper presents a molecular signature of specific and non
specific hybridization with potential consequences for gene expression
analysis. We analyzed the signal intensities of perfect match (PM) and mismatch
(MM) probes of GeneChip microarrays to specify the effect of specific and non
specific hybridization. We found that these events give rise to different
relations between the PM and MM intensities as function of the middle base of
the PMs, namely a triplet- (C>G=T>A>0) and a duplet-like (C=T>0>G=A) pattern of
the PM-MM log-intensity difference upon binding of specific and non specific
RNA fragments, respectively. The systematic behaviour of the intensity
difference can be rationalized on the level of base pairings of DNA/RNA
oligonucleotide duplexes in the middle of the probe sequence. Non-specific
binding is characterized by the reversal of the central Watson Crick (WC)
pairing for each PM/MM probe pair, whereas specific binding refers to the
combination of a WC and a self complementary (SC) pairing in PM and MM probes,
respectively. The intensity of complementary MM introduces a systematic source
of variation which decreases the precision of expression measures based on the
MM intensities
Globally optimal stitching of tiled 3D microscopic image acquisitions
Motivation: Modern anatomical and developmental studies often require high-resolution imaging of large specimens in three dimensions (3D). Confocal microscopy produces high-resolution 3D images, but is limited by a relatively small field of view compared with the size of large biological specimens. Therefore, motorized stages that move the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow direct reconstruction (Stitching) of the whole image from individual image stacks
An automated workflow for parallel processing of large multiview SPIM recordings
Multiview light sheet fluorescence microscopy (LSFM) allows to image
developing organisms in 3D at unprecedented temporal resolution over long
periods of time. The resulting massive amounts of raw image data requires
extensive processing interactively via dedicated graphical user interface (GUI)
applications. The consecutive processing steps can be easily automated and the
individual time points can be processed independently, which lends itself to
trivial parallelization on a high performance cluster (HPC). Here we introduce
an automated workflow for processing large multiview, multi-channel,
multi-illumination time-lapse LSFM data on a single workstation or in parallel
on a HPC. The pipeline relies on snakemake to resolve dependencies among
consecutive processing steps and can be easily adapted to any cluster
environment for processing LSFM data in a fraction of the time required to
collect it.Comment: 13 pages with supplement, LATEX; 1 table, 1 figure, 2 supplementary
figures, 2 supplementary lists, 2 supplementary tables; corrected error in
results table, results unchange
Base pair interactions and hybridization isotherms of matched and mismatched oligonucleotide probes on microarrays
The lack of specificity in microarray experiments due to non-specific
hybridization raises a serious problem for the analysis of microarray data
because the residual chemical background intensity is not related to the
expression degree of the gene of interest. We analyzed the concentration
dependence of the signal intensity of perfect match (PM) and mismatch (MM)
probes in terms using a microscopic binding model using a combination of mean
hybridization isotherms and single base related affinity terms. The signal
intensities of the PM and MM probes and their difference are assessed with
regard to their sensitivity, specificity and resolution for gene expression
measures. The presented theory implies the refinement of existing algorithms of
probe level analysis to correct microarray data for non-specific background
intensities.Comment: 32 pages, 12 figures, 3 table
OpenSPIM - an open access platform for light sheet microscopy
Light sheet microscopy promises to revolutionize developmental biology by
enabling live in toto imaging of entire embryos with minimal phototoxicity. We
present detailed instructions for building a compact and customizable Selective
Plane Illumination Microscopy (SPIM) system. The integrated OpenSPIM hardware
and software platform is shared with the scientific community through a public
website, thereby making light sheet microscopy accessible for widespread use
and optimization to various applications.Comment: 7 pages, 3 figures, 6 supplementary videos, submitted to Nature
Methods, associated public website http://openspim.or
MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules
Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present MTrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. MTrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. The application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. This helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios.V.K. was supported by the IRI Life Sciences postdoc fellowship in
the labs of S.R. and S.P. C.H. and S.R. acknowledge funding by the IRI Life Sciences (Humboldt-Universität zu
Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra “Crossing Boundaries:
Molecular Interactions in Malaria”, which is co-funded by a grant from the Deutsche Forschungsgemeinschaf
(DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. S.P.
was supported by the MDC Berlin
Efficient Bayesian-based Multi-View Deconvolution
Light sheet fluorescence microscopy is able to image large specimen with high
resolution by imaging the sam- ples from multiple angles. Multi-view
deconvolution can significantly improve the resolution and contrast of the
images, but its application has been limited due to the large size of the
datasets. Here we present a Bayesian- based derivation of multi-view
deconvolution that drastically improves the convergence time and provide a fast
implementation utilizing graphics hardware.Comment: 48 pages, 20 figures, 1 table, under review at Nature Method
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