Molecular markers
a technology of molecular markers and markers, applied in the field of gene sequences, can solve the problems of false sense of security, unwarranted and ineffective treatment and/or unnecessary patient anxiety, and process can be quite slow
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example 1
Tissue Collection and RNA Isolation
[0064]Biopsies were collected from patients with squamous carcinoma of the cervix. Punch biopsies with tischler forceps were taken from the tumor lesion for histo-pathological assessment as well as for RNA analysis. For histo-pathological analysis, the tissues were fixed in 10% buffered formalin and processed into paraffin blocks. Multiple step sections were made and stained with hematoxylin and eosin. Where indicated, PAS with and without diastase digestion and mucicarmine stains were performed. Tissues collected for RNA analysis were snapped frozen in liquid nitrogen. Wherever possible, normal cervical tissues far away from the tumor areas were taken for controls. The staging of cancer was done according to recommendations by the International Federation of Obstetrics and Gynacology (FIGO) [FIGO News, 1987). To avoid repetition in evaluating gene expression in the same patient, different patients were included in different studies. In total, 38 c...
example 2
cDNA Cloning and Sequencing
[0067]Re-amplified cDNA fragments were cloned into either the pCR2.1 vector using the TA cloning system from Invitrogen (San Diego, Calif.) or the pCR-TRAP Vector from GenHunter (GenHunter Corp. Nashville, Tenn.). DNA sequencing was performed either using Sequenase Kit Version 2.0 (United States Biochemical, Cleveland, Ohio), or by automated sequencing using ABI Prism 377 DNA Sequencer (Perkin-Elmer Corp., Norwalk, Conn.). The nucleotide sequences obtained were analyzed by BLAST search (National Centre for Biotechnology Information).
example 3
cDNA Microarray Fabrication and Hybridization
[0068]cDNA clones were amplified in 100 μl PCR reactions, 5 μl PCR product was analyzed on a 2% w / v agarose gel after which the remaining PCR products were purified by isopropanol precipitation, resuspended in 15 μl 3×SSC and printed onto poly-L-lysine (Sigma Microsystems Inc., Woburn, Mass.). Housekeeping genes including G3PDH, β-actin, β-2-microglobulin, α-tubulin, cyclophilin and ubiquitin were also spotted as internal controls for normalization. Each slide was hydrated for 2-3 sec over a steaming 100° C. water-bath, snap-dried for 5 see on a 100° C. heating block and crosslinked with 550 mJ ultraviolet irradiation using a Stratalinker (Stratagene, LaJolla, Calif.). The slide was then washed for 10 min in 0.2% w / v SDS and rinsed 5× in distilled water before being denatured for 5 min in 100° C. distilled water, desiccated for 5 min in 95% v / v ethanol and air-dried for 5 min in an 80° C. oven.
[0069]Comparative hybridizations were carried...
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