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101.
102.
Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression 总被引:1,自引:0,他引:1
Patel OV Suchyta SP Sipkovsky SS Yao J Ireland JJ Coussens PM Smith GW 《Veterinary immunology and immunopathology》2005,105(3-4):331-342
The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material. 相似文献
103.
Zimmerman K Feldman B Robertson J Herring ES Manning T 《Veterinary clinical pathology / American Society for Veterinary Clinical Pathology》2003,32(4):213-217
A 1-year-old spayed female Persian cat with alopecia and weight loss had numerous variably ulcerated dermal nodules. Cytologic examination of an aspirate of one of the nodules revealed pyogranulomatous inflammation along with septate hyphae and basophilic round bodies, 0.5–1.0 μm in diameter, surrounded by a thin clear halo (arthrospores). The cytologic diagnosis was dermatophytic pseudomycetoma. Histologically, there were dermal granulomas containing poorly staining, septate hyphae with bulbous spores embedded within abundant amorphous eosinophilic material (Splendore-Hoeppli reaction), and the histologic diagnosis was pseudomycetoma-associated chronic multifocal severe granulomatous dermatitis with lymphocytic perifolliculitis and furunculosis. Microsporum canis was cultured from the lesion. Pseudomycetomas are distinguished from fungal mycetomas, or eumycotic mycetomas, by the findings of multiple lesions, lack of a history of skin trauma, an association with dermatophytes, most commonly Microsporum canis , and, histologically, lack of true cement material and a more abundant Splendore-Hoeppli reaction in pseudomycetomas. Additionally, pseudomycetomas differ from dermatophytosis, in which lesions are restricted to epidermal structures. Persian cats have a high incidence of pseudomycetoma formation, suggesting a heritable predisposition. The prognosis is fair with systemic antifungal therapy. When examining cytologic specimens from Persian cats with single or multiple dermal nodules, especially if pyogranulomatous inflammation is present, a diagnosis of pseudomycetoma should be suspected and is warranted if arthrospores and refractile septate hyphae are present. 相似文献
104.
Bodetti TJ Viggers K Warren K Swan R Conaghty S Sims C Timms P 《Veterinary microbiology》2003,96(2):177-187
The Chlamydiales are a unique order of intracellular bacterial pathogens that cause significant disease of birds and animals, including humans. The recent development of a Chlamydiales-specific 16S rDNA polymerase chain reaction (PCR) assay has enabled the identification of Chlamydiales DNA from an increasing range of hosts and environmental sources. Whereas the Australian marsupial, the koala, has previously been shown to harbour several Chlamydiales types, no other Australian marsupials have been analysed. We therefore used a 16S rDNA PCR assay combined with direct sequencing to determine the presence and genotype of Chlamydiales in five wild Australian mammals (gliders, possums, bilbies, bandicoots, potoroos). We detected eight previously observed Chlamydiales genotypes as well as 10 new Chlamydiales sequences from these five Australian mammals. In addition to PCR analysis we used antigen specific staining and in vitro culture in HEp-2 cell monolayers to confirm some of the identifications. A strong association between ocular PCR positivity and the presence of clinical disease (conjunctivitis, proliferation of the eyelid) was observed in two of the species studied, gliders and bandicoots, whereas little clinical disease was observed in the other animals studied. These findings provide further evidence that novel Chlamydiales infections occur in a wide range of hosts and that, in some of these, the chlamydial infections may contribute to clinical disease. 相似文献
105.
Rachel C. Murray Vet. MB PhD Bridget L. Roberts BVMS Michael C. Schramme Dr. Med. Vet. PhD Sue J. Dyson Vet. MB PhD Marion Branch BSc 《Veterinary radiology & ultrasound》2004,45(2):103-111
Injury of the distal aspect of the deep digital flexor tendon (DDFT) is a recognized cause of lameness, but diagnosis is difficult. This study aimed to improve understanding of DDFT morphology and pathology using retrospective evaluation of magnetic resonance (MR) images. We hypothesized that: (1) The distal aspect of the DDFT in normal horses would have a repeatable proximal/distal pattern and symmetry between limbs and between lobes; (2) DDFT dimensions would be related to bodyweight, navicular bone dimensions and hoof size; (3) this symmetry and pattern would be lost in DDFT injury; and (4) DDFT size would increase with injury. MR images of 64 live horse limbs, 26 with no identified DDFT lesion and 38 with identified DDFT abnormalities, and 19 normal cadaver limbs were analyzed. Using standardized transverse images, measurements of DDFT cross-sectional area, medial-lateral (ML) width and dorsal-palmar depth were obtained at six preselected sites. A uniform distal to proximal shape pattern was identified in all horses. The flattened crescent shape at the insertion changed to a deeper bilobed shape more proximally, with the mid-navicular area having the greatest cross-sectional area. Strong ML (P < 0.0006) and left/right symmetry (P < 0.02) were observed. In addition, there was a strong association between DDFT cross-sectional area and horse weight (P = 0.005) and between DDFT and navicular bone ML width (P = 0.004). Symmetry between sides or between lobes was lost at sites with a unilateral lesion and correlation between horse weight and DDFT cross-sectional area was lost in the presence of lesions. DDFTs with core lesions had a consistent increase in cross-sectional area overall, but other lesion types had no significant increase in size. The shape and symmetry seen in normal tendons could be related to the mechanical demands placed upon individual lobes. The limited increase in cross-sectional area with injury may be explained by the restrictive structures of the hoof, possibly explaining the ongoing pain seen in such lesions. 相似文献
106.
A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation 总被引:1,自引:0,他引:1
Samuel J Harrison Ellie K Mott Kate Parsley Sue Aspinall John C Gray Amanda Cottage 《Plant methods》2006,2(1):19-7
Background
The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants. 相似文献107.
Downing S Chien MB Kass PH Moore PE London CA 《American journal of veterinary research》2002,63(12):1718-1723
OBJECTIVE: To determine the prevalence of activating internal tandem duplications (ITDs) in exons 11 and 12 of c-kit in mast cell tumors (MCTs) of dogs and to correlate these mutations with prognosis. SAMPLE POPULATION: 157 formalin-fixed, paraffin-embedded MCTs from dogs in the pathology database of the Veterinary Medical Teaching Hospital at the University of California, Davis. PROCEDURE: Genomic DNA was isolated from tumor specimens and a polymerase chain reaction procedure was performed to determine whether there were ITDs in exons 11 and 12. RESULTS: We identified ITDs in 1 of 12 (8%) grade-I, 42 of 119 (35%) grade-lI, and 9 of 26 (35%) grade-ll tumors (overall prevalence, 52 of 157 [33%]). Logistic regression analysis revealed that the odds of grade-II and -III tumors possessing an ITD were approximately 5 times greater than that for grade-I tumors, although these odds did not differ significantly. Although MCTs possessing an ITD were twice as likely to recur after excision and twice as likely to result in metastasis as those without an ITD, these values also did not differ significantly. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide evidence that ITDs in c-kit occur frequently in MCTs of dogs. The high prevalence of c-kit activating mutations in MCTs of dogs combined with the relative abundance of mast cell disease in dogs provide an ideal naturally developing tumor in which to test the safety and efficacy of novel small-molecule kinase inhibitors such as imatinib mesylate. 相似文献
108.
109.
1. The effects of reserpine on plasma glucose, FFA and cholesterol and on adrenal cholesterol were determined in 1‐ and 21‐d‐old chicks over a 24‐h period.
2. Irrespective of age, reserpinised chicks became hyperglycaemic and hypocholesteraemic, their stores of adrenal cholesterol were depleted and the mobilisation of the lipid was impaired. The response of the older bird was quantitatively larger than that of the younger bird.
3. Propranolol prevented the increase in plasma glucose concentration seen 1 h after reserpine but it did not affect subsequent changes.
4. It is concluded that reserpine acts as a stressor and it is shown that its effects have not been entirely overcome after 24 h. 相似文献
110.
Leonardo F.C. Brito DVM PhD Patricia L. Sertich MS VMD Keith Durkin BSc Bhanu P. Chowdhary BVScAH PhD Regina M. Turner VMD PhD Lauren M. Greene VMD Sue McDonnell PhD 《Journal of Equine Veterinary Science》2008,28(7):431-436
A 19-month-old Standardbred colt was donated to the University of Pennsylvania School of Veterinary Medicine with a suspicion of intersexuality. The anal−genital distance and penis were normal, and there was no evidence of intersexuality, but the colt was bilaterally cryptorchid. Several aspects of the colt's behavior appeared unusual, including general temperament and behavior described as sympathetically dull and affable. With herd mates, the colt appeared slow to perceive or to learn the usual intraspecies social cues. An atypical gait characterized by intermittent unnatural shuffle of the hind limbs, sliding them along in short rhythmic strides for 3 to 10 seconds at a time was noted at times when a horse might normally transition from a slow walk to a fast walk or a slow trot. Occasionally the colt exhibited slight protrusion of the tongue through the teeth and lips with jaw movements and smacking of the tongue against the teeth as if struggling to retract the tongue to the normal position. Evaluation of the karyotype combined with fluorescent in situ hybridization (FISH) revealed an abnormal male karyotype showing trisomy of chromosome 27 (65, XY + 27). The colt was euthanized at 24 months of age, and a necropsy revealed no significant abnormalities. This case of trisomy was not associated with developmental abnormalities described in other rare reports of trisomy in horses; however, some features were strikingly similar to that of humans with trisomy 21. FISH was demonstrated to be an excellent method for correct identification of equine chromosomes. 相似文献