Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

     

mRNA and microRNA quality control for RT-qPCR analysis

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency.RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.     

Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer

The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18–25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.     

Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus.

The aim of the present study was to test the influence of obesity and the presence of type 2 diabetes mellitus (T2DM) on the expression of ten housekeeping genes and of the 18S rRNA in a group of human adipose tissue samples from the omental and subcutaneous depot. Adipose tissue biopsies were obtained by laparoscopic surgery from lean and obese patients. After the extraction, mRNA levels in adipose tissue samples were quantified by real-time PCR using the commercial HUMAN ENDOGENOUS CONTROL PLATES. From the genes analyzed, 18S rRNA exhibited the most stable expression levels in both depots regardless of the pathophysiological conditions of obesity and obesity-associated T2DM. Contrarily, GAPD was the gene with the highest variation in its expression levels, being upregulated (8.0-fold) in the obese group and downregulated (3.5-fold) in obesity-associated T2DM. Our results show that 18S rRNA may be the most suitable gene for normalization in expression studies performed in human adipose tissue samples obtained from patients suffering from obesity and/or obesity-associated T2DM, whereas GAPD is less appropriate for comparison purposes under these circumstances.     

Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied by elevated levels of plasma visfatin. Recombinant human IL-6 infusion to mimic the exercise-induced IL-6 response (n = 6) had no effect on visfatin mRNA expression in adipose tissue compared with the effect of placebo infusion (n = 6). The finding that exercise enhances subcutaneous adipose tissue visfatin mRNA expression suggests that visfatin has a local metabolic role in the recovery period following exercise.     

mRNA and microRNA selection for breast cancer molecular subtype stratification using meta-heuristic based algorithms

Cancer subtype stratification, which may help to make a better decision in treating cancerous patients, is one of the most crucial and challenging problems in cancer studies. To this end, various computational methods such as Feature selection, which enhances the accuracy of the classification and is an NP-Hard problem, have been proposed. However, the performance of the applied methods is still low and can be increased by the state-of-the-art and efficient methods. We used 11 efficient and popular meta-heuristic algorithms including WCC, LCA, GA, PSO, ACO, ICA, LA, HTS, FOA, DSOS and CUK along with SVM classifier to stratify human breast cancer molecular subtypes using mRNA and micro-RNA expression data. The applied algorithms select 186 mRNAs and 116 miRNAs out of 9692 mRNAs and 489 miRNAs, respectively. Although some of the selected mRNAs and miRNAs are common in different algorithms results, six miRNAs including miR-190b, miR-18a, miR-301a, miR-34c-5p, miR-18b, and miR-129-5p were selected by equal or more than three different algorithms. Further, six mRNAs, including HAUS6, LAMA2, TSPAN33, PLEKHM3, GFRA3, and DCBLD2, were chosen through two different algorithms. We have reported these miRNAs and mRNAs as important diagnostic biomarkers to the stratification of breast cancer subtypes. By investigating the literature, it is also observed that most of our reported mRNAs and miRNAs have been proposed and introduced as biomarkers in cancer subtypes stratification.     

A novel and universal method for microRNA RT-qPCR data normalization

Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.     

A comparison of normalization techniques for microRNA microarray data

Normalization of expression levels applied to microarray data can help in reducing measurement error. Different methods, including cyclic loess, quantile normalization and median or mean normalization, have been utilized to normalize microarray data. Although there is considerable literature regarding normalization techniques for mRNA microarray data, there are no publications comparing normalization techniques for microRNA (miRNA) microarray data, which are subject to similar sources of measurement error. In this paper, we compare the performance of cyclic loess, quantile normalization, median normalization and no normalization for a single-color microRNA microarray dataset. We show that the quantile normalization method works best in reducing differences in miRNA expression values for replicate tissue samples. By showing that the total mean squared error are lowest across almost all 36 investigated tissue samples, we are assured that the bias correction provided by quantile normalization is not outweighed by additional error variance that can arise from a more complex normalization method. Furthermore, we show that quantile normalization does not achieve these results by compression of scale.     

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.     

Ghrelin regulates adiposity in white adipose tissue and UCP1 mRNA expression in brown adipose tissue in mice

To examine the involvement of ghrelin in obesity, we investigated the effects of treatment with peripherally administered ghrelin on food intake, adiposity, and expression of uncoupling protein (UCP) mRNA in brown (BAT) and white (WAT) adipose tissue in mice. Acute bolus administration of ghrelin at a dose of 120 nmol/kg increased cumulative food intake over 4 and 24 h as compared to controls (p<0.05 for each), whereas 12 nmol/kg/day ghrelin showed no remarkable effect (p>0.1). Chronic repeated treatment with 12 nmol/kg/day ghrelin for 7 days increased body weight and adiposity assessed by the weight of adipose tissue, triglyceride content in WAT (p<0.05 for each versus control). In addition, the same treatment decreased and increased mRNA expression of BAT UCP1 and WAT UCP2, respectively (p<0.05 for each). In conclusion, ghrelin can regulate body weight, adiposity and UCPs mRNA expression in mice. The present results provide evidence for a new regulatory loop involving ghrelin and UCP, and add novel insights into the regulatory mechanisms of obesity.     

Physiological activation of brown adipose tissue destabilizes thermogenin mRNA

The amount of mRNA coding for the brown fat specific uncoupling protein thermogenin was followed in the brown adipose tissue of adult mice. As expected, cold exposure or norepinephrine injection caused an increase in the amount of thermogenin mRNA. However, contrary to expectation, the half-life of thermogenin mRNA was dramatically reduced, from about 18 h to about 3 h, when the mice were cold exposed. This destabilization of thermogenin mRNA was not related to the activity of protein synthesis. It was concluded that in brown adipose tissue an unusual mechanism operates which leads to a destabilization of thermogenin mRNA under the same physiological conditions which increase thermogenin gene expression.     

Reference gene selection for quantitative real-time PCR normalization: Application in the caprine mammary gland

In dairy animals, gene expression analysis has become increasing key to understand the biological processes occurring in mammary gland development that shape future milk potential. Selecting high-stability reference genes is crucial to interpret real-time qPCR data. This study investigated the expression stability of five top-ranked candidate reference genes in the goat mammary gland through three assays comparing different experimental conditions (physiological states, sample types and experimental treatments). The expression stability of genes including β-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA, cyclophilin A and ribosomal protein large P0 was analyzed. Normalization for each experimental condition expression data revealed a different reference gene. Nevertheless, in our various assays, genes encoding for ribosomal proteins, 18S rRNA and RPLP0 presented the best expression stability. This result has been confirmed using a combined analysis of stability on the three assays. All genes showed the same distribution within and among the three assays and a different distribution between Ct variability and GeNorm normalization. In addition, the application on Catenin B1 expression using an inappropriate reference gene confirmed erroneous variations in interpretation. To conclude, there is no single ideal reference gene for caprine mammary gland studies and we recommend using a panel of top-ranked reference genes, including RPLP0, at the beginning of each experiment to validate the most stable(s) gene(s).     

Insulin signalling in human adipose tissue

Adipose tissue is a critical regulator of energy balance and substrate metabolism, and synthesizes several different substances with endocrine or paracrine functions, which regulate the overall energetic homeostasis. An excessive amount of adipose tissue has been associated with the development of type 2 diabetes, premature atherosclerosis, and cardiovascular disease. It is believed that the adverse metabolic impact of visceral fat relies on a relative resistance to the action of insulin in this depot compared to other adipose tissue depots. However, information on insulin signalling reactions in human fat is limited. In this paper, we review the major insulin signalling pathways in adipocytes and their relevance for metabolic regulation, and discuss recent data indicating different signalling properties of visceral fat as compared to other fat depots, which may explain the metabolic and hormonal specificity of this fat tissue depot in humans.     

A quantitative map of human primary microRNA processing sites

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Castration-induced changes in microRNA expression profiles in subcutaneous adipose tissue of male pigs

MicroRNAs (miRNAs) are class of molecular regulators found to participate in numerous biological processes, such as adipogenesis and obesity in mammals. To determine the roles of miRNAs involved in castration-induced body fatness, we investigated the different miRNA expression patterns in subcutaneous adipose tissue between intact and castrated male pigs. Our results showed that castration led to decrease serum testosterone but increase serum Leptin levels (P?<?0.01). Moreover, castration also increased adipocyte size, body fat content and backfat thickness in male pigs (P?<?0.01). Meanwhile, miRNA expression profiles in adipose tissue were changed by castration, and 18 miRNAs were considered as the differentially expressed candidates between intact and castrated male pigs. Furthermore, functional analysis indicated that the differential expressed miRNAs and their target genes are involved in the regulation of fatty acid metabolism. In brief, our present study provides a comprehensive view on how miRNAs works in subcutaneous adipose tissue with castration. These results suggested that miRNAs might play an important role in the castration-induced fat deposition in male pigs.