Real-time PCR quantification of precursor and mature microRNA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.     

Normalization of miRNA qPCR high-throughput data: a comparison of methods

Low-density quantitative real-time PCR (qPCR) arrays are often used to profile expression patterns of microRNAs in various biological milieus. To achieve accurate analysis of expression of miRNAs, non-biological sources of variation in data should be removed through precise normalization of data. We have systematically compared the performance of 19 normalization methods on different subsets of a real miRNA qPCR dataset that covers 40 human tissues. After robustly modeling the mean squared error (MSE) in normalized data, we demonstrate lower variability between replicates is achieved using various methods not applied to high-throughput miRNA qPCR data yet. Normalization methods that use splines or wavelets smoothing to estimate and remove Cq dependent non-linearity between pairs of samples best reduced the MSE of differences in Cq values of replicate samples. These methods also retained between-group variability in different subsets of the dataset.     

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs

The serology-based methods routinely used in forensic casework for the identification of biological fluids are costly in terms of time and sample and have varying degrees of sensitivity and specificity. Recently, the use of a molecular genetics-based approach using messenger RNA (mRNA) profiling has been proposed to supplant conventional methods for body fluid identification. However, the size of the amplification products used in these mRNA assays (∼ 200-300 nt) might not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. Recently, there has been an explosion of interest in a novel class of small noncoding RNAs, microRNAs (miRNAs, ∼20-25 bases in length), with numerous published studies reporting that some miRNAs are expressed in a tissue-specific manner. In this article, we provide the first comprehensive evaluation of miRNA expression in dried, forensically relevant biological fluids—blood, semen, saliva, vaginal secretions, and menstrual blood—in an attempt to identify putative body fluid-specific miRNAs. Most of the 452 human miRNAs tested (∼67% of the known miRNAome) were either expressed in multiple body fluids or not expressed at all. Nevertheless, we have identified a panel of nine miRNAs—miR451, miR16, miR135b, miR10b, miR658, miR205, miR124a, miR372, and miR412—that are differentially expressed to such a degree as to permit the identification of the body fluid origin of forensic biological stains using as little as 50 pg of total RNA. The miRNA-based body fluid identification assays were highly specific because the miRNA expression profile for each body fluid was different from that obtained from 21 human tissues. The results of this study provide an initial indication that miRNA profiling may provide a promising alternative approach to body fluid identification for forensic casework.     

Analysis of microRNA signatures using size-coded ligation-mediated PCR

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.     

220-plex microRNA expression profile of a single cell

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.     

Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.     

MicroRNAs in the shoot apical meristem of soybean

Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shoot apical meristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.     

A comprehensive study of multiple mapping and feature selection for correction strategy in the analysis of small RNAs from SOLiD sequencing

High-throughput sequencing is a powerful tool for discovering and profiling microRNAs (miRNAs) to gain further insights into their biogenesis and function. Due to shorter size, short RNAs from deep sequencing dataset are prone to map to multiple loci with an equal number of mismatches, especially among multicopy miRNA precursors and homologous miRNA genes. Systematic analysis of SOLiD sequencing dataset showed that 37.94% short RNAs could simultaneously map to more than one miRNA precursor, and more short RNAs were found to have multiple genomic loci. Improper selection from candidate loci might lose some mapping information, influence miRNA expression profile or even mislead to identify novel miRNAs. A comprehensive study indicated several potential features for correction strategy: location and distribution of mismatches, quality values, expression profiles of multiple isomiRs (miRNA variants), miRNA* and moRs (miRNA-offset-RNAs) at candidate locus and in its flank sequence. Further studies should develop an approach to correct the widespread phenomenon of multiple mapping based on these features, and improve accuracy of profiling and discovering miRNAs.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

Profiling of Circulating MicroRNAs after a Bout of Acute Resistance Exercise in Humans

Recent studies have revealed a new aspect of physiological regulation in which microRNAs (miRNAs) play fundamental roles in diverse biological and pathological processes. Furthermore, it was recently discovered that miRNAs are stably secreted into blood and that circulating miRNAs may play important roles in cell–cell communication. Here, we examined whether the circulating miRNA profile is affected by acute resistance exercise. Twelve males performed a resistance exercise session (bench press and leg press), consisting of five sets of 10 repetitions at 70% of maximum strength, with a 1 min rest between sets. Blood samples were taken before exercise, and at 0 and 60 min, 1 day, and 3 days after exercise. The circulating miRNA profile was determined by microarray analysis. Quantitative real-time PCR confirmed that the miR-149* level increased three days after resistance exercise. In contrast, the miR-146a and miR-221 levels decreased three days after resistance exercise. Our findings suggest that circulating miRNA levels change in response to acute resistance exercise, and miRNAs may play important roles in resistance-exercise-induced adaptation.     

A wide repertoire of miRNA binding sites: prediction and functional implications

MOTIVATION: Over the past decade, deciphering the roles of microRNAs (miRNAs) has relied heavily upon the identification of their targets. Most of the targets that were computationally and experimentally characterized were evolutionarily conserved 'seed' targets, containing a perfect 6-8 nt match between the miRNA 5(')-region and the messenger RNA (mRNA). Gradually, it has become evident that other types of miRNA binding can confer target regulation, but their characterization has been lagging behind. RESULTS: Here, we complement the putative evolutionarily-conserved seed-containing targets by a wide repertoire of putative targets exhibiting a variety of miRNA binding patterns, predicted by our algorithm RepTar. These include non-conserved sites, 'seed' binding sites with G:U-wobbles within the seed, '3(') compensatory' sites and 'centered' sites. Apart from the centered sites, we demonstrate the functionality of these sites and characterize the target profile of a miRNA by the types of binding sites predicted in its target 3(') UTRs. We find that different miRNAs have individual target profiles, with some more inclined to seed binding and others more inclined to binding through 3(') compensatory sites. This diversity in targeting patterns is also evident within several miRNA families (defined by common seed sequences), leading to divergence in the target sets of members of the same family. The prediction of non-conventional miRNA targets is also beneficial in the search for targets of the non-conserved viral miRNAs. Analyzing the cellular targets of viral miRNAs, we show that viral miRNAs use various binding patterns to exploit cellular miRNA binding sites and suggest roles for these targets in virus-host interactions.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.