Ovary Tumor Markers And Methods Of Use Thereof
a technology for ovarian cancer and markers, applied in the field of ovarian tumor markers, can solve the problems of insufficient specificity and sensitivity of most biomarkers commonly used in clinical practice, insufficient specificity and sensitivity to unambiguously distinguish ovarian cancer, and insufficient specificity to be used to screen all women
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example 1
n of Recombinant Human Protein Antigens and Antibodies to Identify Tumor Markers
[0178]Methods
[0179]The entire coding region or suitable fragments of the genes encoding the target proteins were designed for cloning and expression using bioinformatic tools with the human genome sequence as template (Lindskog M et al (2005). Where present, the leader sequence for secretion was replaced with the ATG codon to drive the expression of the recombinant proteins in the cytoplasm of E. coli. For cloning, genes were PCR-amplified from templates derived from Mammalian Gene Collection (http: / / mgc.nci.nih.gov / ) clones or from cDNAs mixtures generated from pools of total RNA derived from Human testis, Human placenta, Human bone marrow, Human fetal brain, using specific primers. Clonings were designed so as to fuse a 10 histidine tag sequence at the 5′ end, annealed to in house developed vectors, derivatives of vector pSP73 (Promega) adapted for the T4 ligation independent cloning method (Nucleic Ac...
example 2
ofiling by Immune-Histochemistry
[0200]Methods
[0201]The analysis of the antibodies' capability to recognize their target proteins in tumor samples was carried out by Tissue Micro Array (TMA), a miniaturized immuno-histochemistry technology suitable for HTP analysis that allows to analyse the antibody immuno-reactivity simultaneously on different tissue samples immobilized on a microscope slide.
[0202]Since the TMAs include both tumor and healthy tissues, the specificity of the antibodies for the tumors can be immediately appreciated. The use of this technology, differently from approaches based on transcription profile, has the important advantage of giving a first hand evaluation on the potential of the markers in clinics. Conversely, since mRNA levels not always correlate with protein levels (approx. 50% correlation), studies based on transcription profile do not provide solid information regarding the expression of protein markers.
[0203]A tissue microarray was prepared containing f...
example 3
n of Target Proteins in Transfected Mammalian Cells
[0210]Methods
[0211]The specificity of the antibodies for each target proteins was assessed by western blot and / or confocal microscopy analysis of eukaryotic cells transiently transfected with a plasmid construct containing the complete sequence of the gene encoding the target proteins. Examples of this type of confocal microscopy experiments are represented for SLC39A10 (corresponding to Transcript ID ENST00000359634), KLRG2 (two cloned sequences corresponding to Transcripts ENST00000340940 and ENST00000393039, corresponding to two transcript variants), ERMP1 (cloned sequence corresponding to Transcripts ENST00000339450).
[0212]For cloning, cDNA were generated from pools of total RNA derived from Human testis, Human placenta, Human bone marrow, Human fetal brain, in reverse transcription reactions and the entire coding regions were PCR-amplified with specific primers pairs. PCR products were cloned into plasmid pcDNA3 (Invitrogen). H...
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