The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
26 Related JoVE Articles!
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
Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics
Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1
). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.
Plant Biology, Issue 64, Genetics, Bioinformatics, Metabolomics, Plant metabolism, Transcriptome analysis, Functional annotation, Computational biology, Plant biology, Theoretical biology, Spectroscopy and structural analysis
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
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
RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Institutions: Arizona State University , Norwegian University of Life Sciences.
This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.
RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg
) and ultraspiracle (usp
), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.
The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.
Neuroscience, Issue 77, Genetics, Behavior, Neurobiology, Molecular Biology, Chemistry, Biochemistry, biology (general), genetics (animal and plant), animal biology, RNA interference, RNAi, double stranded RNA, dsRNA, double gene knockdown, vitellogenin gene, vg, ultraspiracle gene, usp, vitellogenin protein, Vg, ultraspiracle protein, USP, green fluorescence protein, GFP, gustatory perception, proboscis extension response, PER, honey bees, Apis mellifera, animal model, assay
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs
-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf
transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf
animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1
. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf
fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters
Institutions: Princeton University, Princeton University, California Institute of Technology.
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1
promoter upstream of a URA3
cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3
, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
Genetics, Issue 81, transcription, transcription factors, artificial transcription factors, zinc fingers, Zif268, synthetic biology
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
Institutions: Charité - Universitätsmedizin Berlin, Free University Berlin, Charité - Universitätsmedizin Berlin, Charité - Universitätsmedizin Berlin.
Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+
CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+
CSCs and CD133-
bulk, which can be cultivated and functionally analyzed thereafter.
Cancer Biology, Issue 73, Medicine, Stem Cell Biology, Cellular Biology, Molecular Biology, Biomedical Engineering, Genetics, Oncology, Primary cell culture, melanoma, MACS, cancer stem cells, CD133, cancer, prostate cancer cells, melanoma, stem cells, cell culture, personalized treatment
Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery
Institutions: Beckman Research Institute of City of Hope, Beckman Research Institute of City of Hope, Beckman Research Institute of City of Hope.
The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery.
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).
Immunology, Issue 52, SELEX (Systematic Evolution of Ligands by EXponential enrichment), RNA aptamer, HIV-1 gp120, RNAi (RNA interference), siRNA (small interfering RNA), cell-type specific delivery
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
Institutions: Agency for Science, Technology and Research, Singapore, A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, National University of Singapore, Singapore.
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12
. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13
. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6
. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Genetics, Issue 62, ChIP, ChIA-PET, Chromatin Interactions, Genomics, Next-Generation Sequencing
Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Institutions: Joint Unit Hospices de Lyon-bioMérieux, BioMérieux, Hospices Civils de Lyon, Lyon 1 University, BioMérieux, Hospices Civils de Lyon, Hospices Civils de Lyon.
The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1
. ‘How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2
or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4
. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g.
PCA3 in prostate cancer5,6
and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10
. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1
Medicine, Issue 81, Cancer Biology, Genetics, Molecular Biology, Prostate, Retroviridae, Biomarkers, Pharmacological, Tumor Markers, Biological, Prostatectomy, Microarray Analysis, Gene Expression, Diagnosis, Human Endogenous Retroviruses, HERV, microarray, Transcriptome, prostate cancer, Affymetrix
Metabolic Profile Analysis of Zebrafish Embryos
Institutions: School of Medicine, Deakin University.
A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo
during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo
in this relevant vertebrate animal model.
Developmental Biology, Issue 71, Genetics, Biochemistry, Cellular Biology, Molecular Biology, Physiology, Embryology, Metabolism, Metabolomics, metabolic profile, respiration, mitochondria, ATP, development, Oil Red O staining, zebrafish, Danio rerio, animal model
Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Institutions: Indiana University School of Medicine, Indiana University School of Medicine.
Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3
. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6
. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15
. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1
). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17
Medicine, Issue 64, Physiology, beta cell, gene expression, islet, diabetes, insulin secretion, proliferation, adenovirus, rat
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
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
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster
Institutions: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Istituto Italiano di Tecnologia.
The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster
whole embryos, manually dissected larval/adult tissues or in vitro
cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.
Molecular Biology, Issue 90, Northern blotting, Noncoding RNAs, microRNAs, rasiRNA, Gene expression, Gcm/Glide, Drosophila melanogaster
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Purification of Transcripts and Metabolites from Drosophila Heads
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila
as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila
are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
Genetics, Issue 73, Biochemistry, Molecular Biology, Neurobiology, Neuroscience, Bioengineering, Cellular Biology, Anatomy, Neurodegenerative Diseases, Biological Assay, Drosophila, fruit fly, head separation, purification, mRNA, RNA, cDNA, DNA, transcripts, metabolites, replicates, SCA3, neurodegeneration, NMR, gene expression, animal model
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
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
Biochemical Titration of Glycogen In vitro
Institutions: University of Nice - Sophia Antipolis.
Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2
) versus hypoxia (1% O2
). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1
Basic Protocol, Issue 81, Glycogen, Glucoamylase, Fluorescence, Oxidation, Periodic Acid Shiff staining (PAS)
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
Institutions: Bio-Rad Laboratories.
The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.
Cellular Biology, Issue 38, Cell Counting, Gene Silencing, siRNA, Namalwa Cells, IL4, Gene Expression, Electroporation, Real Time PCR
Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
Institutions: AntiCancer, Inc..
There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.
Cellular Biology, Issue 13, Springer Protocols, hair follicles, liposomes, adenovirus, genes, stem cells