-
Jacek K Nowak,
Robert Gromadka,
Marek Juszczuk,
Maria Jerka-Dziadosz,
Kamila Maliszewska,
Marie-Hélène Mucchielli,
Jean-François Gout,
Olivier Arnaiz,
Nicolas Agier, Thomas Tang,
Lawrence P Aggerbeck,
Jean Cohen,
Hervé Delacroix,
Linda Sperling,
Christopher J Herbert,
Marek Zagulski,
Mireille Bétermier
[show abstract]
[hide abstract]
ABSTRACT: Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.
Eukaryotic Cell 01/2011; 10(3):363-72. · 3.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: There are many sources of variation in dual labelled microarray experiments, including data acquisition and image processing. The final interpretation of experiments strongly relies on the accuracy of the measurement of the signal intensity. For low intensity spots in particular, accurately estimating gene expression variations remains a challenge as signal measurement is, in this case, highly subject to fluctuations.
To evaluate the fluctuations in the fluorescence intensities of spots, we used series of successive scans, at the same settings, of whole genome arrays. We measured the decrease in fluorescence and we evaluated the influence of different parameters (PMT gain, resolution and chemistry of the slide) on the signal variability, at the level of the array as a whole and by intensity interval. Moreover, we assessed the effect of averaging scans on the fluctuations. We found that the extent of photo-bleaching was low and we established that 1) the fluorescence fluctuation is linked to the resolution e.g. it depends on the number of pixels in the spot 2) the fluorescence fluctuation increases as the scanner voltage increases and, moreover, is higher for the red as opposed to the green fluorescence which can introduce bias in the analysis 3) the signal variability is linked to the intensity level, it is higher for low intensities 4) the heterogeneity of the spots and the variability of the signal and the intensity ratios decrease when two or three scans are averaged.
Protocols consisting of two scans, one at low and one at high PMT gains, or multiple scans (ten scans) can introduce bias or be difficult to implement. We found that averaging two, or at most three, acquisitions of microarrays scanned at moderate photomultiplier settings (PMT gain) is sufficient to significantly improve the accuracy (quality) of the data and particularly those for spots having low intensities and we propose this as a general approach. For averaging and precise image alignment at sub-pixel levels we have made a program freely available on our web-site http://bioinfome.cgm.cnrs-gif.fr to facilitate implementation of this approach.
BMC Bioinformatics 04/2009; 10:98. · 2.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two-colour microarrays are widely used to perform transcriptome analysis. In most cases, it appears that the 'red' and 'green' images resulting from the scan of a microarray slide are slightly shifted one with respect to the other. To increase the robustness of the measurement of the fluorescent emission intensities, multiple acquisitions with the same or different PMT gains can be used. In these cases, a systematic correction of image shift is required.
To accurately detect this shift, we first developed an approach using cross-correlation. Second, we evaluated the most appropriate interpolation method to be used to derive the registered image. Then, we quantified the effects of image shifts on spot quality, using two different quality estimators. Finally, we measured the benefits associated with a systematic image registration. In this study, we demonstrate that registering the two images prior to data extraction provides a more reliable estimate of the two colours' ratio and thus increases the accuracy of measurements of variations in gene expression.
http://bioinfome.cgm.cnrs-gif.fr/.
Bioinformatics 11/2007; 23(20):2686-91. · 5.47 Impact Factor
-
Bioinformatics. 01/2007; 23:2686-2691.
