Surgery Intact female Sprague Dawley rats at six, 26 or 52 weeks of age, weighing 154 eleven g, 281 25 g, and 330 30 g respectively, had been anaes thetized with an intraperitoneal injection of ketamine and xylazine as described earlier. The left knee was shaved, scrubbed with Betadine Option, and draped with sterile sheets. A medial incision was made with the knee, the patella was deflected laterally and a one. 0 mm hole was drilled in to the inter condylar notch. An intramedullary rod was placed retrograde into the left femur. The incision was closed with wound clips. A closed uncomplicated transverse mid diaphyseal femoral fracture was induced with a Bonnarens and Einhorn device. Ran domly selected rats from among individuals scheduled for sur gery have been made use of for 0 time no fracture sham controls. Rats have been euthanized at 0, 0.

four, 1, two, four, and 6 weeks soon after frac ture to get a complete of six time factors at every single from the three ages. Six rats per time point per age group scientific assays have been picked for micro array analysis. Radiographs have been manufactured at fracture, at 1 week after fracture, and at euthanasia. The femora have been swiftly harvested, and 1 third in the fem oral length, centered over the fracture site, was collected. This contained the fracture callus with related cortical bone and marrow and was frozen in liquid nitrogen and stored at 75 C. RNA Sample Planning and Microarray Processing Samples were ready as described inside the Affymetrix GeneChip Expression Examination Technical Manual. The sam ple planning is described right here in quick. Total RNA was extracted through the tissue by TRIzol with disruption from the tissue in the Brinkman Polytron homogenizer.

RNA from two rats from the exact same age and time point was pooled for each microar ray sample. Samples with thirty g RNA were purified on RNeasy columns by Qiagen and then converted to double stranded cDNA having a Superscript Double Stranded cDNA Synthesis Kit. The cDNA was then expressed as biotin labeled cRNA by in vitro tran scription using the Enzo RNA Transcript reference 2 Labeling Kit. Just about every sample was spiked with bioB, bioC, bioD, and cre. The biotin labeled cRNA was fragmented non enzymatically. The fragmented cRNA was hybridized to 54 Rat U34A microarrays from the Affymetrix hybridization buffer for 16 hours at 45 C. The hybridized arrays have been washed and stained in the Affymetrix Fluidics Station 400 to attach fluorescent labels for the biotin, fol lowed by biotin labeled antibody, after which a second staining with fluorescent labeling of the biotin.

Just about every array was scanned twice through the Agilent GeneArray Scanner G2500A. 3 arrays from three independent samples have been finished for each age at each time level. Information Analysis The Rat U34A GeneChip Microarray has probe sets for in excess of eight,700 rat genes. Most probe sets have twenty various probes for that identical gene on each and every array with 20 supplemental mismatch controls. The data were analyzed with Affyme trix Microarray Suite five. 0 and Affymetrix Data Mining Instrument 3. 0 software. Microarray Suite was applied to scale the mRNA expression of all genes to an regular of 500 for each array. For each gene, the software package reported a sig nal value plus a Present Marginal Absent get in touch with.

This latter algorithm was a statistical comparison of the variation amongst the several probe sets for every gene in contrast towards the noise level and gave a get in touch with for each gene as Current, Marginal, or Absent. The plan then in contrast the sig nal worth of each gene in the fractured samples towards the signal worth with the same gene from the unfractured manage sample. The main difference in between the two signal amounts, rela tive to your variability involving the several probes for each gene, yielded a probability of alter because of opportunity alone. Genes with p less than 0. 005 have been judged significantly dif ferent from your same gene during the unfractured sample. This much more conservative p value was employed to reduce false positive responses.