Ovarian Expression Profile of KISS1 and NGF Genes in Indian Goat

Background: Litter size is one of the most important economic traits that translates into higher returns for farmers. KISS 1 and NGF genes plays significant role in female reproduction. The purpose of the study was to evaluate the potential variation in the of expression levels of KISS1 and NGF genes in ovarian tissue of two different goat breeds with diverse prolificacy. Methods: Phenotypic records pertaining to litter size of Barbari and Jamunapari goats were collected. Total RNA was extracted from both breeds' ovarian tissues for real-time PCR quantification of the KISS1 and NGF genes, using GAPDH and β-actin as endogenous controls. The target gene’s expression levels were measured and the fold-change was determined. Result: The phenotypic recording revealed that Barbari goats are more prolific in nature than Jamunapari goats. The expression levels of KISS1 and NGF genes were higher in Barbari goat and lower in Jamunapari goat (P less than 0.05). The results of this study can be used to further discover the crucial role of KISS1 and NGF genes in reproduction for improving the prolificacy in goats.

Circulating microRNA alternations in primary hyperuricemia and gout

Objectives MicroRNAs (miRNAs) are short single-stranded RNAs that play a role in the post-transcriptional regulation of gene expression. Their deregulation can be associated with various diseases, such as cancer, neurodegenerative, and immune-related diseases. The aim of our study was to compare miRNA levels in plasma that could potentially influence the progression of hyperuricemia to gout, since the mechanism of progression is still unclear. Methods Total RNA, including miRNA, was isolated from the plasma of 45 patients with asymptomatic hyperuricemia, 131 patients with primary gout (including 16 patients having a gout attack), and 130 normouricemic controls. The expression of 18 selected miRNAs (cel-miR-39 and cel-miR-54 as spike-in controls, hsa-miR-16-5p and hsa-miR-25-3p as endogenous controls, hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30a-3p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-133a-3p, hsa-miR-142-3p, hsa-miR-143-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, hsa-miR-223-3p, hsa-miR-488-3p and hsa-miR-920) was measured using qPCR. Results We found that hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-142-3p, and hsa-miR-223-3p were significantly upregulated (p < 0.001) in the plasma of hyperuricemia and gout patients compared to normouricemic individuals. As part of the follow-up of our previous study, we found a negative correlation between hsa-miR-17-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, and hsa-miR-223-3p with plasma levels of chemokine MCP-1. Additionally, we found a positive correlation between CRP and plasma levels of hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, and hsa-miR-223-3p. Five of those miRNAs (hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-222-3p) also had a positive correlation with serum creatinine and therefore a negative correlation with eGFR. Conclusion Five miRNAs were significantly upregulated in the plasma of patients with hyperuricemia and gout (and those during a gout attack) compared to normouricemic controls. We also found a correlation between the plasma levels of several miRNA and plasma levels of MCP-1, CRP, serum creatinine, and eGFR.

Description of a CSF-Enriched miRNA Panel for the Study of Neurological Diseases

Background: The study of circulating miRNAs in CSF has gained tremendous attention during the last years, as these molecules might be promising candidates to be used as biomarkers and provide new insights into the disease pathology of neurological disorders. Objective: The main aim of this study was to describe an OpenArray panel of CSF-enriched miRNAs to offer a suitable tool to identify and characterize new molecular signatures in different neurological diseases. Methods: Two hundred and fifteen human miRNAs were selected to be included in the panel, and their expression and abundance in CSF samples were analyzed. In addition, their stability was studied in order to propose suitable endogenous controls for CSF miRNA studies. Results: miR-143-3p and miR-23a-3p were detected in all CSF samples, while another 80 miRNAs were detected in at least 70% of samples. miR-770-5p was the most abundant miRNA in CSF, presenting the lowest mean Cq value. In addition, miR-26b-5p, miR-335-5p and miR-92b-3p were the most stable miRNAs and could be suitable endogenous normalizers for CSF miRNA studies. Conclusions: These OpenArray plates might be a suitable and efficient tool to identify and characterize new molecular signatures in different neurological diseases and would improve the yield of miRNA detection in CSF.

Selection of the Most Stable Endogenous Control Genes for Microrna Quantitation in Chicken Ovarian Follicles

MicroRNAs (miRNAs or miRs) belong to a class of small non-coding RNAs of 19 to 24 nucleotides long that act as negative gene regulators at the post-transcriptional level. Quantitative PCR (q-PCR) is a commonly used technique in the profiling of miRs, and identification of reliable endogenous controls is crucial for proper data normalisation. To date, no study has been performed on reference miRs for the normalisation of miR expression in chicken ovarian tissues. Therefore, the aim of the present study was to experimentally identify the most stable reference mirs for normalisation of miR q-PCR expression data in the chicken ovary. Relying on high-throughput sequencing, five putative reference miR (let-7a-3p, miR-140a-3p, miR-22-5p, miR-33-5p, miR-99a-3p) were identified and subsequently analysed in a total of 66 tissue samples. The stability of candidate endogenous controls validated by the most widely used algorithms, geNorm, NormFinder, and BestKeeper, showed that let-7a-3p, miR-140a-3p, and miR-22-5p are the most appropriate choice of reference genes. Application of different normalisation approaches to the relative quantitation of randomly chosen miR-1552-5p in chicken ovarian follicles indicated the impact of the selected reference genes on miR expression. Further, the results revealed a downregulation of miR-1552-5p. In summary, the three identified endogenous reference miRs are suitable for profiling the miR expression in ovarian tissues of laying hens. Our findings provide valuable information for future miR expression studies in the avian ovary.

Identification of suitable controls for miRNA quantification in T-cells and whole blood cells in sepsis

Complex immune dysregulation is a hallmark of sepsis. The occurring phases of immunosuppression and hyperinflammation require rapid detection and close monitoring. Reliable tools to monitor patient’s immune status are yet missing. Currently, microRNAs are being discussed as promising new biomarkers in sepsis. However, no suitable internal control for normalization of miRNA expression by qPCR has been validated so far, thus hampering their potential benefit. We here present the first evaluation of endogenous controls for miRNA analysis in human sepsis. Novel candidate reference miRNAs were identified via miRNA microArray. TaqMan qPCR assays were performed to evaluate these microRNAs in T-cells and whole blood cells of sepsis patients and healthy controls in two independent cohorts. In T-cells, U48 and miR-320 proved suitable as endogenous controls, while in whole blood cells, U44 and miR-942 provided best stability values for normalization of miRNA quantification. Commonly used snRNA U6 exhibited worst stability in all sample groups. The identified internal controls have been prospectively validated in independent cohorts. The critical importance of housekeeping gene selection is emphasized by exemplary quantification of imuno-miR-150 in sepsis patients. Use of appropriate internal controls could facilitate research on miRNA-based biomarker-use and might even improve treatment strategies in the future.

Identification of Endogenous Control miRNAs for RT-qPCR in T-Cell Acute Lymphoblastic Leukemia

Optimal endogenous controls enable reliable normalization of microRNA (miRNA) expression in reverse-transcription quantitative PCR (RT-qPCR). This is particularly important when miRNAs are considered as candidate diagnostic or prognostic biomarkers. Universal endogenous controls are lacking, thus candidate normalizers must be evaluated individually for each experiment. Here we present a strategy that we applied to the identification of optimal control miRNAs for RT-qPCR profiling of miRNA expression in T-cell acute lymphoblastic leukemia (T-ALL) and in normal cells of T-lineage. First, using NormFinder for an iterative analysis of miRNA stability in our miRNA-seq data, we established the number of control miRNAs to be used in RT-qPCR. Then, we identified optimal control miRNAs by a comprehensive analysis of miRNA stability in miRNA-seq data and in RT-qPCR by analysis of RT-qPCR amplification efficiency and expression across a variety of T-lineage samples and T-ALL cell line culture conditions. We then showed the utility of the combination of three miRNAs as endogenous normalizers (hsa-miR-16-5p, hsa-miR-25-3p, and hsa-let-7a-5p). These miRNAs might serve as first-line candidate endogenous controls for RT-qPCR analysis of miRNAs in different types of T-lineage samples: T-ALL patient samples, T-ALL cell lines, normal immature thymocytes, and mature T-lymphocytes. The strategy we present is universal and can be transferred to other RT-qPCR experiments.