Transcriptome / Gene expression
Transcriptome / Gene expression
Since 1999, Skuldtech has provided gene expression profiling services through open system methods (Digital Gene Expression, SAGE) associated with sequencing/pyrosequencing and proprietary bioinformatics tools. This high throughput platform is dedicated to the development and acquisition of genomic data through the analysis of gene expression and accurate gene identification and quantification processes. This tool can be used for identifying all the genes induced in each physiopathological situation in an exhaustive and sensitive manner. However, the genes are either already known or not and the genome of the organism is either sequenced or not.
All the results can be implemented in an interactive database and can be compared with thousands of gene profile libraries, which have been gathered in a unique relationship database called Biotag. This database is an active and user-friendly website access tool, which enables to use and analyze "Digital Gene Expression" (SAGE) gene profiles. Thus, each user can easily search and compare the gene profiles he/she has selected, compare transcriptome libraries, design biomarker sets in a specific tissue and/or tumor, anticipate drug effects on complete organism, etc.
Biotag covers different vegetal and animal organisms as well as H. sapiens, M. musculus, and model organisms such as D. melanogaster, C. elegans, or A. thaliana.
Publications
- D Berthier, I Chantal, S Thevenon, H Sakande, JC Maillard, Z Bengaly, D Piquemal, J Marti, G Cuny. Study of bovine trypanotolerance by whole transcriptome analysis. Ann N Y Acad Sci. 2008 Dec;1149:71-6.
- J de Lorgeril, Y Gueguen, C Goarant, E Goyard, C Mugnier, J Fievet, D Piquemal D and Bachère E (2008) A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection. Mol Immunol. 16
- Y Gueguen, J Garnier, L Robert, MP Lefranc, I Mougenot, J de Lorgeril, M Janech, PS Gross, GW Warr, B Cuthbertson, MA Barracco, P Bulet, A Aumelas, Y Yang, D Bo, J Xiang, A Tassanakajon, D Piquemal D and E Bachere E (2006) PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature. Dev Comp Immunol. 30:283-8.
- JC Maillard, D Berthier, S Thevenon, D Piquemal, I Chantal, J Marti. Efficiency and limits of the Serial Analysis of Gene Expression (SAGE) method: discussions based on first results in bovine trypanotolerance. Vet Immunol Immunopathol. 2005 Oct 18;108(1-2):59-69.
- D Berthier, R Quéré, S Thevenon, D Belemsaga, D Piquemal, J Marti, JC Maillard. Serial analysis of gene expression (SAGE) in bovine trypanotolerance: preliminary results. Genet Sel Evol. 2003;35 Suppl 1:S35-47.
After the development of genomic probe diagnostic systems in the 1990's, followed by PCR-based systems, Skuldtech has developed and patented the Mini-Array method that allows one-step multiple detections. This method has been developed to allow the accessibility of powerful array technology.
Within Mini-Array technology, hybridization time is reduced to 15 min, whereas other methods require a longer hybridization time. Hybridization of the PCR product on a nylon membrane and revelation of the hybrids by an antibody increase considerably the ability of pathogen's detection.
The Mini-Array method is based on a detection combining target nucleic acid amplification by PCR and a specific hybridization evidenced by a colorimetric reaction on a nylon membrane. Detection of the hybrids is done using enzyme-linked antibodies producing a dark-blue precipitate with a substrate.
This method exhibits numerous advantages compared the other molecular diagnostic tools such as hybridization or amplification by PCR: possibility of a multiple detection, higher sensitivity, high specificity, no questionable results, etc. It is ease to use and result interpretation could be done by nacked eye, its reliability and its high sensitivity would replace advantageously the regular and tedious methods of evidencing the amplification results by electrophoresis, and especially eliminating the use of Ethidium bromide which is a mutagenic reagent.
Multiple detections are one of the major advantages of the Mini-Array. In case of multiple detections, multiple sets of specific primers are used, and the different amplicons issued from the PCR hybridize with the homologous probes plotted in pre-defined positions on the nylon membrane. By the way, the interpretation of the results visualized on the membrane is easy and can be done by the naked eye. An internal control is added to each reaction. It is a PCR and hybridization positive control DNA fragment which is used to prevent false negatives due to experimental errors.
This technology increases substantially the power detection limit of pathogens and is a highly sensitive method contributing to virus prophylaxis:
- 10 000 times more sensitive than the Dot Blot method.
- 100 times more sensitive than standard detection by electrophoresis of PCR amplified products.
It is the ideal method for detection of low level or early infections in view to limit the risks of horizontal and vertical transmissions. Its reliability and high sensitivity can replace advantageously the regular and tedious methods of evidencing the amplification results by electrophoresis, and particularly eliminating the use of Ethidium bromide which is a carcinogenic dye.
The test is rapid: one to 48 reactions can be done in parallel in only 4 - 5 hours.
The membranes provided with the kit are ready to use. The interpretation of the results is easy: presence or absence of blue dots can be detected by naked eye. Little equipment is required: except for the thermocycler, only basic equipment such as a water-bath, a small bench centrifuge and a shaker are necessary. Thus, this method can be easily used in farms or hatcheries already using PCR as a diagnostic tool.
Publications
The technology patented by Skuldtech has been presented in a scientific publication and scientific press:
- Journal of Virological Methods,
- Advocate Magazine,
- Aquatic Magazine.
