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Although a large number of small RNAs (sRNAs) have been discovered, it is very likely that the screens conducted so far have not reached saturation. Recently, many methods for predicting and identifying new sRNAs have been developed. However, it remains unclear what the total number of sRNAs within a genome is and how many types of sRNAs exist in plants and animals. In this article, combined methods...
Distinct classes of small RNAs, 20–32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable...
MicroRNAs (miRNAs) are small (∼22nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating...
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...
Epigenetics in general and microRNA (miRNA) in particular are an important and growing field of research, and while significant advances in the role of miRNA in a variety of diseases including cancer have been made, the majority of the information on the relationship between miRNA and its putative target proteins have been made on homogenized tissue, which, while useful, provides no information on...
Small RNAs are widespread in plants and animals. They largely include microRNAs (miRNAs) and short interfering RNAs (siRNAs), and they play key roles in gene and chromatin regulations. Here we describe in detail the method for an effective construction of the recently developed short tandem target mimic (STTM) technology to block small RNA functions in plants and animals. STTM is a powerful technology...
MicroRNA (miRNA) variants that share the sequences with other closely related miRNAs have been identified by deep sequencing and have been implicated in the diverse regulation of their target genes. The miRNA variants that originate from the same miRNA precursor are among the most common and have been termed “isomiRs.” IsomiRs can be generated by several mechanisms such as differential processing...
The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20–30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present...
miRNAs are short (20–23nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable...
microRNAs are important regulators of gene expression that guide translational repression and degradation of target mRNAs. Only relatively few miRNA targets have been characterized, and computational prediction is hampered by the relatively small number of nucleotides that seem to be involved in target recognition. Argonaute (Ago) crosslinking and immunoprecipitation (CLIP) in combination with next-generation...
The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step...
MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21–23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs).The high relative stability of miRNA in common clinical tissues and biofluids...
Using both high-throughput sequencing and real-time PCR, the miRNA transcriptome can be analyzed in complementary ways. We describe the necessary bioinformatics pipeline, including software tools, and key methodological steps in the process, such as adapter removal, read mapping, normalization, and multiple testing issues for biomarker identification. The methods are exemplified by the analysis of...
The abundance of miRNAs – small non-coding RNAs involved in posttranscriptional regulation of gene expression – in tissues and body fluids of cancer patients hold great promise to identify specific biomarkers, which may be useful for early diagnosis as well as to predict the clinical outcome and treatment response. For the extraction and quantification of miRNAs from cells and tissues, present technologies...
Colorectal cancer (CRC) develops and progresses through a systematic selection for (epi) genetic alterations that drive the transformation from normal colon epithelium to adenocarcinoma. These changes affect both noncoding RNAs and mRNAs and so define the clinical behaviour of cancer cells within a distinctive host genetic and environmental context. Although earlier diagnosis and more effective treatment...
The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. Past approaches using in silico methods and reporter constructs lack biological context that may enhance or inhibit target recognition. To address these limitations, several labs have utilized crosslinking and immunoprecipitation (CLIP) of Argonaute (Ago) proteins to identify miRNA targets...
MicroRNAs (miRNAs) are ∼21nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years...
miRNA is known to regulate up to several hundreds coding genes, thus the integrated analysis of miRNA and mRNA expression data is an important problem. Unfortunately, the integrated analysis is challenging since it needs to consider expression data of two different types, miRNA and mRNA, and target relationship between miRNA and mRNA is not clear, especially when microarray data is used. Fortunately,...
We quantify the strength of miRNA–target interactions with MIRZA, a recently introduced biophysical model. We show that computationally predicted energies of interaction correlate strongly with the energies of interaction estimated from biochemical measurements of Michaelis–Menten constants. We further show that the accuracy of the MIRZA model can be improved taking into account recently emerged experimental...
Exosomes are RNA and protein-containing nanovesicles secreted by all cell types and found in abundance in body fluids, including blood, urine and cerebrospinal fluid. These vesicles seem to be a perfect source of biomarkers, as their cargo largely reflects the content of parental cells, and exosomes originating from all organs can be obtained from circulation through minimally invasive or non-invasive...
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