Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.
17 Related JoVE Articles!
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3
), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).
Genetics, Issue 62, Target ID, miRNA, ncRNA, RNAi, genomics
Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR
Institutions: LSU Health Sciences Center, University of Milan.
MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.
Medicine, Issue 83, microRNAs, biomarkers, miRNA profiling, qPCR, cerebrospinal fluid, RNA, DNA
Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes
Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAM
construct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.
Developmental Biology, Issue 98, Clinical grade neural stem cells, miRNA profiling and effects, in vitro differentiation, three dimensional culture
Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays
Institutions: University Hospital Basel, University Hospital Tübingen.
Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called “cancer stem cells” (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro
is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro
. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.
Medicine, Issue 95, Cancer stem cells, spheres assay, ovarian, single cell, SOX2, in vitro assay, ovarian carcinoma
Electrochemotherapy of Tumours
Institutions: Institute of Oncology Ljubljana, University of Ljubljana.
Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring.
Medicine, Issue 22, electrochemotherapy, electroporation, cisplatin, bleomycin, malignant tumours, cutaneous lesions
In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells
Institutions: National Cancer Institute.
Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.
Medicine, Issue 96, Ovarian cancer, tumor-initiating cells, cancer stem cell, tumorigenicity, spheroid, mouse, gynecological cancer
Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles
Institutions: University of Edinburgh, University of Edinburgh.
The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro
, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro
. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.
Cellular Biology, Issue 97, reproductive biology, ovary, culture technique, follicle, oocyte, thecal cell, immunocytochemistry
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Institutions: The University of Sydney, The University of Sydney.
Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g.
TaqMan Array Card) and nanofluidics arrays (e.g.
OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.
Molecular Biology, Issue 98, microRNA, ncRNA, probe-based assays, high-throughput PCR, Nanofluidics / Open Arrays, reverse-transcription, pre-amplification, qPCR
Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines
Institutions: Indiana University School of Medicine.
Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.
Medicine, Issue 91, cancer stem cells, stem cell markers, ovarian cancer, chemoresistance, cisplatin, cancer progression
MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium
Institutions: JBSA Fort Sam Houston.
The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.
Molecular Biology, Issue 88, microRNA, microarray, human induced-pluripotent stem cells, retinal pigmented epithelium
Isolation of Small Noncoding RNAs from Human Serum
Institutions: University of Technology, Sydney, University of Technology, Sydney, Royal Prince Alfred Hospital.
The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1
. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues.
Bioengineering, Issue 88, small noncoding RNA isolation, microRNAs, human serum, qPCR, guanidinium thiocyanate , Phase Lock Gels, arrays
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media
Institutions: Drexel University College of Medicine.
Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.
Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media
Genetics, Issue 76, Molecular Biology, Cellular Biology, Medicine, Biochemistry, Genomics, Pharmacology, Exosomes, RNA, MicroRNAs, Biomarkers, Pharmacological, Exosomes, microRNA, qPCR, PCR, blood, biomarker, TLDA, profiling, sequencing, cell culture
MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)
Institutions: University of Toronto, Sunnybrook Health Sciences Centre, Toronto, Canada, Sunnybrook Health Sciences Centre, Toronto, Canada, Sunnybrook Research Institute.
MicroRNAs (miRNAs) are single-stranded, 18–24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1
, and cell cycle regulation3
. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4
by binding to their target messenger RNAs (mRNAs)5
. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6
. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7
. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8
Quantification of miRNA levels in prostate tumor samples
. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1
). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences.
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.
Cancer Biology, Issue 63, Medicine, cancer, primer assay, Prostate, microRNA, tumor, qPCR
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
Institutions: SwitchGear Genomics.
MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.
Genetics, Issue 55, MicroRNA, miRNA, mimic, Clone, 3' UTR, Assay, vector, LightSwitch, luciferase, co-transfection, 3'UTR REPORTER, mirna target, microrna target, reporter, GoClone, Reporter construct
Bacterial Delivery of RNAi Effectors: Transkingdom RNAi
Institutions: Charité Campus Mitte.
RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tk
RNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli
, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tk
RNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv
locus from Yersinia pseudotuberculosis
that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA
gene from Listeria monocytogenes
, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli
strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli
. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tk
RNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.
Microbiology, Issue 42, Transkingdom RNAi, shRNA, gene therapy, cancer, multidrug resistance, bacteria
Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay
Institutions: Arizona State University, Arizona State University.
Luminescent Identification of Functional Elements in 3’UTRs (3’LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3’UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3’LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3’LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3’UTRs cloned in 96-well plates, and two test miRNAs, let-7c
. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.
Molecular Biology, Issue 99, microRNA, luciferase assay, 3' untranslated region, high-throughput, transfection, post-transcriptional gene regulation, cancer