Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas

Background/Objective: Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods: Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization

Referenz-miRNAs für die miRNAome Analyse des Urothelkarzinoms

Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods: Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.Die quantitative reverse Transkriptions-Polymerase-Kettenreaktion (RT-qPCR) ist eine häufig verwendete Methode zur Untersuchung von microRNA(miRNA)-Expressionen bei Tumorerkrankungen. Die relative Quantifizierung der gemessenen miRNAs, basierend auf endogenen Referenzgenen, ist dabei unerlässlich, um die Variabilität, bedingt durch die einzelnen Teilschritte der Analyse, zu kompensieren. Eine Literaturrecherche ergab, dass bis zum jetzigen Zeitpunkt keine Studien zur Ermittlung von Referenz-miRNAs für das Harnblasenkarzinom vorliegen. Das Ziel dieser Studie war es, in systematischer Weise geeignete Referenz-miRNAs für RT-qPCR basierte miRNA- Expressionsstudien des Harnblasenkarzinoms zu identifizieren. Methodik: Unter Zuhilfenahme eines miRNA-Microarrays wurden aus insgesamt 24 Karzinom- und Normalgewebeproben der Harnblase Kandidaten von Referenz-miRNAs anhand ihrer Invarianz in der Expressionsstabilität zwischen den Proben ermittelt. Die Validierung dieser potenziellen Referenz-miRNAs erfolgte zusammen mit den häufig in der Literatur verwendeten small RNAs, RNU6B, RNU48 und Z30, an 58 Gewebeproben mittels RTqPCR. Die anschließende bioinformatorische Analyse wurde mit den Computerprogrammen geNorm, NormFinder und BestKeeper durchgeführt. Grundlegende Ergebnisse: Insgesamt wurden 16 potenzielle Referenz-miRNAs auf der Grundlage der miRNAMicroarraydaten identifiziert. Nach der Validierung mittels RT-qPCR zeigten miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR- 424, miR-874, RNU6B, RNU48 und Z30 keine Unterschiede zwischen den Gewebeproben, sodass ihre Eignung als Referenzgene mit den drei Programmen ermittelt werden konnte. Daraus resultierten unterschiedliche Referenzgenkombinationen. Schlussfolgerungen: Die vorliegende Studie lieferte die erste systematische Analyse zur Identifizierung geeigneter Referenz-miRNAs für miRNA- Expressionsstudien des Harnblasenkarzinoms mittels RT-qPCR. Verschiedene Referenzgenkombinationen ergaben sowohl für starkals auch für schwach- regulierte miRNAs vergleichbare Expressionsergebnisse. Besonders eindrucksvoll konnte die fehlerhafte Normalisierung mit der RNU6B belegt werden, die bisher am häufigsten in miRNA-Studien als Referenzgen zum Einsatz kam. Die Kombination aus vier (miR-101, miR-125a-5p, miR-148b und miR-151-5p) bzw. aus drei (miR-148b, miR-181b und miR-874) Referenz-miRNAs wird für die Normalisierung von Expressionsstudien beim Harnblasenkarzinom empfohlen

Functional Epigenetic Analysis of Prostate Carcinoma: A Role for Seryl-tRNA Synthetase?

Transcriptional silencing, as a result of aberrant promoter hypermethylation, is a common mechanism through which genes in cancer cells become inactive. Functional epigenetic screens using demethylating agents to reexpress transcriptional silenced genes may identify such inactivated genes for needing further evaluation. We aimed to identify genes so far not known to be inactivated by promoter hypermethylation in prostate cancer. DU-145 and LNCaP cells were treated with the DNMT inhibitor zebularine. Expression changes of total RNA from treated and untreated cells were compared using an RNA expression microarray. Genes upregulated more than 2-fold were evaluated by RT-qPCR in 50 cases of paired normal and tumor tissues of prostate cancer patients. SARS was found to be downregulated in prostate cancer in 42/50 cases (84%). In addition, GADD45A and SPRY4 showed a remarkable diminished expression (88% and 74%, resp.). The gold standard for promoter hypermethylation-inactivated genes in prostate cancer (GSTP1) was repressed in 90% of our patient samples. ROC analyses reported statistically significant AUC curves in SARS, GADD45A, and GSTP1 and positive Spearman correlations were found between these genes. SARS was discovered to be a novel gene that is repressed in prostate cancer and could therefore be recommended for its involvement in prostate carcinogenesis

Diagnostic and Prognostic Potential of MicroRNA Maturation Regulators Drosha, AGO1 and AGO2 in Urothelial Carcinomas of the Bladder

Bladder cancer still requires improvements in diagnosis and prognosis, because many of the cases will recur and/or metastasize with bad outcomes. Despite ongoing research on bladder biomarkers, the clinicopathological impact and diagnostic function of miRNA maturation regulators Drosha and Argonaute proteins AGO1 and AGO2 in urothelial bladder carcinoma remain unclear. Therefore, we conducted immunohistochemical investigations of a tissue microarray composed of 112 urothelial bladder carcinomas from therapy-naïve patients who underwent radical cystectomy or transurethral resection and compared the staining signal with adjacent normal bladder tissue. The correlations of protein expression of Drosha, AGO1 and AGO2 with sex, age, tumor stage, histological grading and overall survival were evaluated in order to identify their diagnostic and prognostic potential in urothelial cancer. Our results show an upregulation of AGO1, AGO2 and Drosha in non-muscle-invasive bladder carcinomas, while there was increased protein expression of only AGO2 in muscle-invasive bladder carcinomas. Moreover, we were able to differentiate between non-muscle-invasive and muscle-invasive bladder carcinoma according to AGO1 and Drosha expression. Finally, despite Drosha being a discriminating factor that can predict the probability of overall survival in the Kaplan–Meier analysis, AGO1 turned out to be independent of all clinicopathological parameters according to Cox regression. In conclusion, we assumed that the miRNA processing factors have clinical relevance as potential diagnostic and prognostic tools for bladder cancer

Milk composition of indian rhinoceros (Rhinoceros unicornis) and changes over lactation

The objective of this study was to determine the major nutrient composition of Indian rhinoceros milk (Rhinoceros unicornis) over the first 13 mo of an 18-mo lactation period and to compare the results to those of previous studies on rhinoceros, African elephant (Loxodonta africana), and horse milk (Equus ferus caballus). The following parameters were measured: dry matter (DM), crude ash (ASH), crude protein (CP), ether extract (EE), nitrogen-free extract (NFE; calculated), lactose, calcium (Ca), phosphorus (P), magnesium (Mg), fatty acids (FAs), and gross energy (GE). DM, ASH, CP, and EE were determined with a proximate analysis, lactose with infrared spectroscopy and an enzymatic method, minerals with an autoanalyzer, FA with gas chromatography, and GE with bomb calorimetry. Milk samples were collected from two Indian rhinoceros cows from Zoo Basel. Rhino A gave birth to her third calf on 10 September 2012; three samples were collected and analyzed (colostrum, milk 1 wk and 2 wk postpartum). Rhino B gave birth to her eighth calf on 05 October 2013; samples were collected and 15 were chosen for the analyses (from colostrum to 13 mo postpartum). The composition of rhino B's colostrum was 13.8% DM (wet-weight basis), 4.8% ASH, 61.8% CP, 0.7% EE, 32.6% NFE, 26.7% lactose, 0.59% Ca, 0.54% P, 0.2% Mg (DM basis), and 20.3 MJ GE/kg DM. Rhino B's sample collected 13 mo postpartum averaged 8.0% DM (wet-weight basis), 3.6% ASH, 16.3% CP, 1.8% EE, 78.3% NFE, 84.7% lactose, 0.54% Ca, 0.48% P, 0.09% Mg (on DM basis), and 17.43 MJ GE/kg DM. The main FAs in rhino B's and rhino A's samples were C10 : 0, C12 : 0, C16 : 0, C18 : 1n9c, and C18 : 2n6c. Milk of the Indian rhinoceros is low in fat and protein but high in lactose, which is comparable to the milk composition of other rhinoceros species and horses, but not African elephants

The Effects of Feed Particle Size and Floor Type on the Growth Performance, GIT Development, and Pododermatitis in Broiler Chickens

The aim of the present study was to evaluate the effects of feed particle size and flooring designs on organ traits, performance and pododermatitis in broilers. A total of 480 broilers (Ross 308) of both sexes were randomly assigned to two feeding groups (finely or coarsely ground pelleted diets; with addition of 5% to 10% intact wheat in coarsely diets) and four different housing systems (litter; litter with floor heating; partially or fully slatted floor) with three subgroups each. A coarse diet increased the final gizzard and pancreas weights (p < 0.001) while decreasing the risk of Isthmus gastrici dilatation compared to a fine diet (p < 0.001). Broilers fed a coarse diet displayed an increased final body weight (p = 0.023) and led to a favourable feed conversion ratio. Final body weight was the highest (p < 0.001) for birds housed on partially or fully slatted floor. Housing birds on litter with floor heating showed the lowest pododermatitis scoring (p < 0.001). It seems to be favourable to use coarse diets for organ development, whereas slatted floors seem to foster enlargement of the Isthmus gastrici. Increasing growth performance was possible both when using coarse diets or slatted floors

Outcome of an Experimental Study in Growing Turkeys Suspected of Having a Diet Related, Uncommon and Uncoordinated Gait

On the occasion of a clinical case on a turkey farm and based on the suspicion that the diet composition could be the cause, an eight-week diagnostic trial was performed with turkey poults (n = 54) divided into two groups (control and experimental). The levels of vitamin A in the starter and grower diets of the control group were 7168 and 5213 IU/kg diet, but <1000 IU/kg in the experimental ones. Vitamin A and uric acid contents were measured in the serum, while liver samples were taken to determine the vitamin A content. Parts of the central nervous system and some internal organs were examined histologically. In the sixth week, ruffled feathers and uncoordinated gait were the earliest signs seen in the experimental group. The vitamin A content in the liver samples significantly decreased in the experimental group (0.09 mg/kg vs. 29.5 mg/kg). The serum level of uric acid in the experimental group was significantly higher (12.8 mg/dL vs. 3.38 mg/dL). Birds in the experimental group showed squamous metaplasia in the oesophagus. No histopathological alterations were seen in the central nervous system. The elevated uric acid level in the serum is worth mentioning