The Five Steps to miRNA Profiling

MicroRNAs (miRNAs) are small, non-coding RNAs that play a role in regulating cancer by acting as both tumor suppressors and oncogenes. Ranging in size from 18–25 nucleotides, miRNAs function in feedback mechanisms to regulate many cellular processes including cell proliferation, apoptosis, cell signaling and tumorigenesis (1).

Not surprisingly, dysregulation of miRNA expression can have serious repercussions. For example, miRNAs are dysregulated in almost all human cancers (1). Because of the potential to influence cancer growth and development, there is growing interest in miRNA profiling to identify possible biomarkers for cancer diagnosis or prognosis, as well as potential therapeutic targets (1).

Growing interest in miRNAs as both biomarkers of disease and therapeutic targets drives the need for fast and effective methods for miRNA profiling. Profiling miRNA targets follows a relatively simple workflow: sample selection, RNA extraction, RNA QC and quantitation, RNA profiling and data analysis (2,3). So what happens at each step?

Five steps of miRNA profiling

The Five Steps of miRNA Profiling

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microRNA: The Small Molecule with a Big Story

Introduction

miR-34 precursor secondary structure. The colors indicate evolutionary conservation. Ppgardne [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

RNA molecules have become a hot topic of research. While I was taught about messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA), many more varieties have come into the nomenclature after I graduated with my science degrees. Even more interesting, these RNAs do not code for a protein, but instead have a role in regulating gene expression. From long non-coding RNA (lncRNA) to short interfering RNA (siRNA), microRNA (miRNA) and small nucleolar RNA (snoRNA), these classes of RNAs affect protein translation, whether by hindering ribosomal binding, targeting mRNA for degradation or even modifying DNA (e.g., methylation). This post will cover the topic of microRNAs, explaining what they are, how researchers understand their function and role in metabolism, cancer and cardiovascular disease, and some of the challenges in miRNA research.

What are microRNAs? MicroRNAs (miRNAs) are short noncoding RNAs 19–25 nucleotides long that play a role in protein expression by regulating translation initiation and degrading mRNA. miRNAs are coded as genes in DNA and transcribed by RNA polymerase as a primary transcript (pri-miRNA) that is hundreds or thousands of nucleotides long. After processing with a double-stranded RNA-specific nuclease, a 70–100 nucleotide hairpin RNA precursor (pre-miRNA) is generated and transported from the nucleus into the cytoplasm. Once in the cytoplasm, the pre-miRNA is cleaved into an 18- to 24-nucleotide duplex by ribonuclease III (Dicer). This cleaved duplex associates with the RNA-induced silencing complex (RISC), and one strand of the miRNA duplex remains with RISC to become the mature miRNA. Continue reading

Announcing: Stem Cells in the 4th Dimension—Mechanisms of Stem Cell Aging and Maturation

Image courtesy of Carlos Marti-Figueroa, Wisconsin Institute for Discovery, University of Wisconsin, Madison

Image courtesy of Carlos Marti-Figueroa, Wisconsin Institute for Discovery, University of Wisconsin, Madison

On April 13th, the BTC Institute and Promega Corporation will host the 11th Annual Wisconsin Stem Cell Symposium — Stem Cells in the 4th Dimension: Mechanisms of Stem Cell Aging and Maturation.

Our co-coordinators at the UW-Madison Stem Cell and Regenerative Medicine Center have put together an outstanding list of presenters, including leading researchers who are investigating the effects of aging on stem cell populations and their progeny and recapitulating aging mechanisms in vitro to mature human stem cell derivatives and transplants.

The morning session will review systemic and cell autonomous factors known to impact stem cell maturation, aging and senescence. The afternoon session will focus on using these approaches and understanding to develop in vitro models of matured, stem cell-derived neural, cardiac, and pancreatic cells and tissues for regenerative medicine applications.

HIGHLIGHTED ISSUES:

  • Endocrine, micro-RNA, epigenetic, and metabolic regulators of aging
  • Systemic regulators elucidated by parabiosis
  • Treatment of age-related stem cell dysfunction
  • In vivo and in vitro models of neural, musculoskeletal, cardiac, and pancreatic tissue maturation

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The Promise of miRNAs as Therapeutic Agents in Treating Disease

When researchers first identified a new family of seemingly non-functional “junk” RNA molecules, it’s unlikely they could have predicted the power and promise of these nucleic acids. The small, non-coding, single-stranded RNAs – typically 21-25 base pairs in length – were first discovered over 20 years ago in C. elegans, yet they were quickly found to be ubiquitous in species from worms to flies to plants to mammals. The role of these novel RNAs in the regulation of developmental pathways in worms, coupled with their prevalence, inspired researchers to better understand their significance.

We now know that miRNAs (for microRNAs) serve as post-transcriptional repressors of gene expression by targeting degradation of mRNA or interfering with mRNA translation. While small, each can have a big effect; a single miRNA can regulate dozens to hundreds of distinct target genes. They’ve been implicated in a variety of critical cellular processes such as differentiation, development, metabolism, signal transduction, apoptosis and proliferation.

Tissue-specific expression patterns revealed that specific miRNAs are enriched in mammalian tissues including adult brain, lung, spleen, liver, kidney and heart.  More compelling was the identification of abnormal miRNA expression in tumorigenic cell lines. It’s no wonder that this growing family quickly became ripe for exploration in disease development.

Basic research on miRNA is making its way into the clinic.

Research on miRNA is making its way into the clinic.

Within only a few years, a rapidly expanding body of research supported the theory that miRNA expression may indeed play a role in the development of human diseases including cardiovascular disease, cancer, diabetes, cystic fibrosis, and liver disease. Investigations into the expression of miRNAs in cardiovascular disease, in particular, have demonstrated not only their value as disease markers, but also how their dysregulation is linked to disease processes.

More recently a new possibility is being explored: can miRNA be manipulated to interfere with disease progression? Continue reading

A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation

Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.

mirnaThe accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.

Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.

Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control. Continue reading

miRNA: An Ancient, Small and Important Gene Regulatory Element

On formation and function of miRNAs.  Used as described by license for Wikimedia Commons.

On formation and function of miRNAs. Used under license for Wikimedia Commons.

MicroRNA (miRNA) is a group of small (approximately 18–24 nucleotide) single-stranded, non-coding RNAs that function in negative regulation of gene expression.

Lest that non-coding part make miRNA sound inconsequential, read on. While discovery of miRNA is relatively recent, miRNA is some ancient and seriously important gene regulatory material.

Identification of miRNA was published in late 1993 by Lee, Feinbaum and Ambros regarding their work with the worm, C. elegans.

miRNA has been studied in plants, mammals and even viruses, where miRNA functions to repress mRNA expression through base-pairing to complementary sequences in mRNA. This binding can silence the mRNA by several mechanisms, including cleavage of the mRNA, shortening of the poly(A) tail and interference with translation efficiency. Continue reading

MicroRNAs as Circulating Biomarkers

12097693_lMicroRNAs (miRNAs) are short strands of RNA averaging between 19-24 nucleotides in length that were first discovered in C.elegans and subsequently shown to exist in species ranging from algae to humans (1). Speculated to be merely “junk” more than a decade ago, miRNAs have emerged as powerful regulators of a wide array of cellular processes because of their influence on gene expression at the posttrancriptional level. Dysregulation of these miRNAs is also associated with life-threatening conditions such as cancer and cardiovascular disease, which points to a potential use of miRNAs in diagnosis and treatment. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, protected from degradation by inclusion in lipid or lipoprotein complexes. This opens up the possibility to exploit miRNA as a useful diagnostic tool in clinical samples. Continue reading

It’s All in the Details: Simplified and Improved miRNA Purification from Tissue

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

Consider the microRNA. At only about 21–26 nucleotides in length, microRNAs (miRNAs) are short, but don’t dismiss miRNAs as too short to accomplish much of anything. miRNAs are a multifunctional workhorse that play a key role in a number of genetic regulatory mechanisms throughout the plant and animal kingdoms and even in certain viruses. Scientists estimate that the human genome encodes about 2,000 different miRNAs and miRNAs account for about 3–4% of human genes (1).

Next Tuesday, we invite you to consider the microRNA with us as we host a webinar discussing the growing field of miRNA research and highlight a new, simplified miRNA purification method. Follow the link below to register.

It’s All in the Details (or Small RNA): Simplified and Improved miRNA Purification from Tissue

Presented by Douglas Horejsh, Ph.D.
Tuesday, January 27

Reference

  1. Valinezhad Orang, A., Safaralizadeh, R. and Kazemzadeh-Bavili, M. (2014) Mechanisms of miRNA-mediated gene regulation from common downregulation to mRNA-specific upregulation. Int. J. Genomics Article ID 970607 http://dx.doi.org/10.1155/2014/970607

How MicroRNAs Have a Big Effect on Genetic Regulation

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

miR-133 microRNA (green) and myogenin mRNA (red) in differentiating C2C12 cells. Image by Ryan Jeffs, courtesy of Wikimedia Commons.

Some of us scientists who have been around for a while still think about RNA molecules falling into three categories: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). However, within the past few decades I have had to revise my outdated RNA classification scheme as scientists discover exciting new classes of RNAs that do some fairly amazing things. For example, in the early 1980s, Thomas Cech discovered ribozymes, RNAs that have catalytic functions (1), and in the early 1990s, researchers began to take interest in short noncoding RNAs that act as a genetic regulators, the first of which was discovered in C. elegans (2). RNA is no longer simply a biological middleman between DNA and protein. These ephemeral nucleic acid molecules play a much bigger role of cellular physiology and gene regulation than we had previously ascribed to RNA.

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Tools for Understanding miRNA Regulation of mRNA Expression: The Vector

7825ma_450pxmicroRNAs  (miRNA) are abundant RNA molecules around 21 nucleotides long that regulate specific mRNA expression by directly interacting with the mRNA molecule. Our understanding of miRNA function in mRNA regulation has grown exponentially as more miRNA molecules have been described. As of 2013, more than 24,000 miRNA molecules had been described from more than 140 separate species, indicating that miRNA regulation is conserved across species. In humans, 2,500 mature miRNAs have been described, and researchers predict that 60% of human protein-coding genes may be targets of miRNA regulation. Most often miRNA regulation of an mRNA results in decreased expression, either by destabilizing the mRNA or by inducing translational repression. Very recently, some researchers have reported up regulation of mRNA through miRNA activity.

Since miRNA molecules are so abundant within cells and across species and their target sequences are found in so many protein-coding genes, understanding how miRNA regulation of mRNAs acts in concert with the many other levels of gene expression regulation becomes a complex, but fundamental, biological question.

To probe miRNA regulation of mRNA, the proper tools and experimental design are essential. Continue reading