Podcasts about total rna

Background: Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to Screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results: Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions: Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

Background: In order to define new prognostic subgroups in patients with glioblastoma a miRNA screen (> 1000 miRNAs) from paraffin tissues followed by a bio-mathematical analysis was performed. Methods: 35 glioblastoma patients treated between 7/2005 - 8/2008 at a single institution with surgery and postoperative radio(chemo) therapy were included in this retrospective analysis. For microarray analysis the febit biochip "Geniom (R) Biochip MPEA homo-sapiens" was used. Total RNA was isolated from FFPE tissue sections and 1100 different miRNAs were analyzed. Results: It was possible to define a distinct miRNA expression pattern allowing for a separation of distinct prognostic subgroups. The defined miRNA pattern was significantly associated with early death versus long-term survival (split at 450 days) (p = 0.01). The pattern and the prognostic power were both independent of the MGMT status. Conclusions: At present, this is the first dataset defining a prognostic role of miRNA expression patterns in patients with glioblastoma. Having defined such a pattern, a prospective validation of this observation is required.

The aim of the present study was to estimate the influence of uterine NOS-system and the sexual steroids estrogen and progesterone on the blood supply of the uterus and the ovaries. Five trotter mares were examined in total during four estrous cycles and one early pregnancy. The investigations contained color Doppler- and ultrasound recordings as soon as the extraction of blood samples and endometrial biopsies. The first cycle was medically uninfluenced and investigations were carried out on Days 0 (= ovulation), 1, 5, 11, 15 and daily during estrous, whereas in estrous only one endometrial biopsy was taken. In the second cycle luteal regression was induced on Day 5 of diestrous with the PGF2α-analogon Tiaprost (Ilirenâ, 450 mg i.m.) and in estrous ovulation was induced with hCG (Choriolutinâ, 2500 IE i.v.), when the size of one follicle reached 35 mm. Investigations were carried out in this cycle immediately before giving PGF2α and 0.5, 1, 3, 6, 12, 24, 36, 48 hours after that and then once a day up to ovulation. Endometrial biopsies were taken in each case before induction of luteolysis, 12 and 36 hours after that, before injection of hCG and immediately after ovulation. During the further two cycles the mares were treated orally with the NO donor ISDN (ISDN 40â, 30 mg, twice a day). Investigations were carried out on Days 0, 1 and 5 of the first cycle (ISDN I), as well as on Days 11, 15 and during estrous of the second cycle (ISDN II). During early pregnancy color Doppler recordings and blood samples were taken on Days 0, 1, 5, 11 and 15 of pregnancy, while endometrial biopsies were only gained on day 15. The analysis of uterine blood flow was based on the time-averaged maximum velocity (TAMV). Estrogens and progesterone were measured by an enzyme immunoassay. Nitrate concentrations were evaluated using Griess reacting. Total RNA was isolated from endo-metrial biopsies. Transcriptes encoding the estrogen receptors a and b and the endothelial as well as the inducible nitric oxide-synthase (eNOS and iNOS) were quantified by RT-PCR-analysis. The expression of eNOS-mRNA in control, as well as in the PGF2a-induced cycle, followed a consistent wavelike pattern (p < 0.05 and p < 0.0001). Both, eNOS and iNOS, showed their maximum of expression especially on Day 5 of diestrous and during estrous, when the highest levels of either progesterone or estrogen were measured. In the PGF2a-induced cycle, a significant connection between eNOS and progesterone was proved (p < 0.05). There was a positive correlation between the expression of the eNOS-mRNA and the uterine blood flow (p < 0.05) in the control cycle. In the PGF2a-induced cycle these two parameters also showed a similar cyclic pattern. The levels of estrogen-receptors in the endometrium or concentrations of estrogen in plasma were not related to the endometrial NO-synthases or to TAMV values. Plasma concentrations of nitrate were independent from the stage of estrous cycle and consequently were proved as unsuitable for the question raised here (in this case). The application of the NO donor ISDN leaded to a marked decrease of the uterine NOS-expression when compared with control cycle (p < 0.0001). An increase of uterine or ovarian perfusion during the application of ISDN was not provable (p > 0.05). In result of the biopsies on day 5 and of simultaneous ISDN application luteolysis was induced next to all mares. On day 15 of pregnancy, an increase in uterine and ovarian perfusion as well as an enhancement in iNOS-mRNA expression was observed. In conclusion, the results of this study indicate that the endometrial NO-synthases play an important role in the regulation of uterine blood supply during estrous cycle and pregnancy of the mare. Progesterone as well as estrogen seem to stimulate the endometrial expression of eNOS and iNOS, by which these hormones may indirectly influence uterine and ovarian blood flow.

Carcinoembryonic antigen (CEA) is a tumor marker that belongs to a family of closely related molecules with variable expression patterns. We have developed sets of oligonucleotide primers for the specific amplification of transcripts from individual CEA-family members using the reverse transcriptase/ polymerase chain reaction (RT/PCR). Specific primer sets were designed for CEA, non-specific cross-reacting antigen (NCA), biliary glycoprotein (BGP), carcinoembryonic antigen gene-family members 1, 6 and 7 (CGMI, CGM6 and CGM7), and one set for all pregnancy-specific glycoprotein (PSG) transcripts. Primers were first tested for their specificity against individual cDNA clones and product-hybridization with internal, transcript-specific oligonucleotides. Total RNA from 12 brain and 63 gynecological tumors were then tested for expression of CEA-related transcripts. None were found in tumors located in the brain, including various mesenchymal and neuro-epithelial tumors. CEA and NCA transcripts were, however, present in an adenocarcinoma located in the nasal sinuses. In ovarian mucinous adenocarcinomas, we always found co-expression of CEA and NCA transcripts, and occasionally BGP mRNA. CEA-related transcripts were also found in some serous, endometrioid and clear-cell ovarian carcinomas. CEA, NCA and BGP transcripts were present in endometrial carcinomas of the uterus and cervical carcinomas, whereas uterine leiomyomas were completely negative. No transcripts were found from CGM 1, CGM6, CGM7 or from PSG genes in any of the tumors tested. The PCR data were compared with immunohistochemical investigations of ovarian tumors at the protein level using CEA (26/3/13)-, NCA-50/90 (9A6FR) and NCA-95 (80H3)-specific monoclonal antibodies.