Digital Gene Expression (SAGE-like method)Biosensor / Mini-Array
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 profiles 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 organisms, 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
1. F Guerfali, D Laouini, L Guizani-Tabbane, F Ottones, K Ben-Aissa, A Benkahla, L Manchon, D Piquemal, S Smandi, O Mghirbi, T Commes, J Marti and K Dellagi (2008) Simultaneous gene expression profiling in human macrophages infected with Leishmania major parasites using SAGE. BMC Genomics 9:238
2. M Koshiji, K Kumamoto, K Morimura, Y Utsumi, M Aizawa, M Hoshino, S Ohki, S Takenoshita, M Costa, T Commes, D Piquemal, CC Harris, KM Tchou-Wong (2007) Correlation of N-myc downstream-regulated gene 1 expression with clinical outcomes of colorectal cancer patients of different race/ethnicity. World J Gastroenterol. 28:2803-10.
3. R Quere, A Baudet, B Cassinat, G Bertrand, J Marti, L Manchon, D Piquemal, C Chomienne, T Commes (2007) Pharmacogenomic analysis of acute promyelocytic leukemia cells highlights CYP26 cytochrome metabolism in differential all-trans retinoic acid sensitivity. Blood 109:4450-60.
4. S Bandyopadhyay, Y Wang, R Zhan, SK Pai, M Watabe, M Iiizumi, E Furuta, S Mohinta, W Liu, S Hirota, S Hosobe, T Tsukada, K Miura, Y Takano, K Saito, T Commes, D Piquemal, T Hai, K Watabe (2006) The tumor metastasis suppressor gene Drg-1 down-regulates the expression of activating transcription factor 3 in prostate cancer. Cancer Res. 15:11983-90.
5. I Mechaly,S Bourane,D Piquemal,M Al-Jumaily,S Venteo,S Puech,F Scamps,J Valmier,P Carroll (2006) Gene profiling during development and after a peripheral nerve traumatism reveals genes specifically induced by injury in dorsal root ganglia. Mol Cell Neurosci. 32:217-29
6. I Guinobert, M Viltard, D Piquemal, JM Elalouf, J Marti and M Lelievre-Pegorier (2006) Identification of Differentially Expressed Genes between Fetal and Adult Mouse Kidney: Candidate Gene in Kidney Development. Nephron Physiol. 102:81-91
7. R Quéré, L Manchon, M Lejeune, O Clément, B Bonafoux, T Commes, D Piquemal and J Marti (2004). Mining SAGE data for antisense polyadenylated transcripts. Nucleic Acids Res. 32:e163
8. R Quere, A Baudet, B Cassinat, G Bertrand, D Piquemal, L Manchon, J Marti,C Chomienne,T Commes (2005) All trans retinoic acid (atRA) differentiation markers in normal and retinoid-resistant acute promyelocytic leukemia cells revealed induction of atRA metabolism as relevant prognostic of APL sensitivity to therapy. Blood 106: 910A-910A 3256
9. A Baudet, L Delva, P Balaguer, D Piquemal, J Marti, T Commes (2005) NCoA4 containing transcriptional complexes modulates response of myelomonocytic leukemic cell line to retinoids and VD3. Blood 106: 140B-140B 4238
10. S Bandyopadhyay, SK Pai, S Hirota, S Hosobe, T Tsukada, K Miura, Y Takano, K Saito, T Commes, D Piquemal, M Watabe, S Gross, Y Wang, J Huggenvik and K Watabe (2004). PTEN Up-Regulates the Tumor Metastasis Suppressor Gene Drg-1 in Prostate and Breast Cancer. Cancer Res. 64:7655-60.
11. B Bonafoux, M Lejeune, D Piquemal, R Quere, P Aguilar-Martinez, J Marti and T Commes (2004). Analysis of remnants reticulocyte mRNA provides new markers of the erythroïd lineage. Haematologica 89: 1430-36
12. E Varlet-Marie, M Audran, M Lejeune, B Bonafoux, M Sicart, J Marti, D Piquemal and T Commes (2004). Real time PCR analysis of human reticulocytes genes reveals altered erythropoiesis: potential use to detect recombinant human erythropoietin doping. Haematologica 89:991-97.
13. S Bandyopadhyay, SK Pai, S Hirota, S Hosobe, Y Takano, K Saito, D Piquemal, T Commes, M Watabe, SC Gross, Y Wang, S Ran, K Watabe (2004). Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression. Oncogene 23:5675-81.
14. J Marti, D Piquemal, L Manchon and T Commes (2002) Transcriptomes for serial analysis of gene expression. J Soc Biol. 196:303-7
15. I Guinobert, M Viltard, D Piquemal, T Commes, C Merlet-Benichou, J Marti, M Lelievre-Pegorier (2002) Characterization of the transcriptome of the fetal kidney. Journal of the American Society of Nephrology 13: 300A-300A Suppl.
16. D Piquemal, T Commes, L Manchon, M Lejeune, C Ferraz, D Pugnère, J Demaille, JM Elalouf and J Marti (2002) Transcriptome analysis of monocytic leukemia cell differentiation. Genomics 80 : 361-372
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.
When using Mini-Array technology, the 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 considerably increase pathogen detection possibilities.
The Mini-Array method is based on a detection process combining target nucleic acid amplification by PCR and a specific hybridization evidenced by a colorimetric reaction on a nylon membrane. The hybrids are detected by using enzyme-linked antibodies producing a dark-blue precipitate with a substrate.
This method provides numerous advantages compared to other molecular diagnostic tools such as hybridization or amplification by PCR. These advantages include the possibility of a multiple detection, a higher sensitivity, a high specificity, no questionable results, etc. It is easy to use and results can be interpreted visually. Due to its reliability and high sensitivity, it can advantageously replace the common and tedious methods employed for evidencing amplification results obtained by electrophoresis. Another advantage is that it enables to avoid using Ethidium bromide which is a mutagenic reagent.
Multiple detections are one of the major advantages of the Mini-Array. In the 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. The interpretation of the results observed on the membrane is easy and can be done by visual inspection. 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 substantially increases the power detection limit of pathogens and is a highly sensitive method contributing to virus prophylaxis as it is:
- 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 solution for detecting low level or early infections in aim of limiting the risks of horizontal and vertical transmissions. Due to its reliability and high sensitivity, it can advantageously replace the common and tedious methods employed for evidencing amplification results obtained by electrophoresis. Another advantage is that it enables to avoid using Ethidium bromide which is a carcinogenic dye.
The test is rapid - one to 48 reactions can be performed at the same time in only 4 - 5 hours.
The membranes provided with the kit are ready to use. The interpretation of the results is easy - the presence or absence of blue dots can be detected visually. 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. Therefore, this method can easily be 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.
