Identification of sequences particularly useful for the diagnosis and identification of therapeutic targets for osteoarthritis
a technology of sequence identification and osteoarthritis, which is applied in the field of identification and selection of sequences, can solve the problems of cartilage degradation and osteoarthritis, significant impact on productivity and or quality of life, and much more disability, and achieve the effect of monitoring the efficacy of the therapeutic regimen and diagnosing the degree of osteoarthritis
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example 1
RNA Extraction, cDNA Library Construction and EST Analysis
[0389]Normal cartilage was obtained from the donor program of Department of Orthopaedics and Rehabilitation, University of Miami. OA cartilage samples were obtained from either areas of very early cartilage degeneration (mild) or from sites of moderate, marked or severe cartilage degeneration during either arthroscopic knee surgery or total knee replacement. OA severity was graded according to the system described by Marshall (Marshall K W. J Rheumatol, 1996:23(4) 582-85). Briefly, each of the six knee articular surfaces was assigned a cartilage grade with points based on the worst lesion seen on each particular surface. Grade 0 is normal (0 points), Grade I cartilage is soft or swollen but the articular surface is intact (1 point). In Grade II lesions, the cartilage surface is not intact but the lesion does not extend down to subchondral bone (2 points). Grade III damage extends to subchondral bone but the bone is neither er...
example 2
Microarray Construction
[0394]Microarrays using ESTs isolated from the four cDNA libraries as described above were created.
[0395]PCR products (˜40 ul) of cDNA clones from OA cartilage cDNA libraries as described above were utilized in the same 96-well tubes used for amplification, are precipitated with 4 ul ( 1 / 10 volume) of 3M sodium acetate (pH 5.2) and 100 ul (2.5 volumes) of ethanol and stored overnight at −20° C. They are then centrifuged at 3,300 rpm at 4° C. for 1 hour. The obtained pellets were washed with 50 ul ice-cold 70% ethanol and centrifuged again for 30 minutes. The pellets are then air-dried and resuspended well in 50% dimethylsulfoxide (DMSO) or 20 ul 3×SSC overnight. The samples are then deposited either singly or in duplicate onto Gamma Amino Propyl Silane (Corning CMT-GAPS or CMT-GAP2, Catalog No. 40003, 40004) or polylysine-coated slides (Sigma Cat. No. P0425) using a robotic GMS 417 or 427 arrayer (Affymetrix, Calif.). The boundaries of the DNA spots on the mic...
example 3
Target Nucleic acid Preparation and Hybridization using Constructed Arrays
[0397]Preparation of Fluorescent DNA Probe from mRNA
[0398]Fluorescently labeled target nucleic acid samples are prepared for analysis with an array of the invention.
[0399]2 μg Oligo-dT primers are annealed to 2 ug of mRNA isolated from a cartilage sample from patient diagnosed with osteoarthritis as described above in a total volume of 15 ul, by heating to 70° C. for 10 min, and cooled on ice. The mRNA is reverse transcribed by incubating the sample at 42° C. for 1.5-2 hours in a 100 μl volume containing a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 25 mM DTT, 25 mM unlabeled dNTPs, 400 units of Superscript 11 (200 U / uL, Gibco BRL), and 15 mM of Cy3 or Cy5 (Amersham). RNA is then degraded by addition of 15 μl of 0.1N NaOH, and incubation at 70° C. for 10 min. The reaction mixture is neutralized by addition of 15 μl of 0.1N HCL, and the volume is brought to 500 μl with TE (10 mM Tris,...
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