Questo lavoro è stato eseguito da studenti universitari a sud-ovest Oklahoma State University, OK Weatherford. Questo lavoro è stato sostenuto da NSF RUI-0963258, NIH OK-INBRE 2P20RR016478, GCAT e OCAST HR09-137S. L&#39;(ok1468) vsm1 mutante è stato isolato dal Consorzio Knockout Gene Oklahoma. Gli autori ringraziano Dan Stefanovic e Tanner Wheeler per il loro aiuto prezioso durante l&#39;esecuzione del progetto.

<ol>
	<li>Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V., Pennetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hVAPB,+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans,+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction.">hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the Neuromuscular Junction.</a> Hum Mol Genet. 17, 266-280 (2008).</li><li>Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved,+secreted,+and+acts+as+a+ligand+for+Eph+receptors.">The Amyotrophic Lateral Sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors.</a> Cell. 133, 963-977 (2008).</li><li>Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., Zamorano, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Active+Zone+Precursor+Vesicles.">Assembly of Active Zone Precursor Vesicles.</a> Journal of Biological Chemistry. 281, 6038-6047 (2005).</li><li>Lustgarten, V., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis.">Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis.</a> Molecular and Cellular Biology. 19, 4480-4494 (1999).</li><li>Vi&#241;uela, A., Snoek, L., Riksen, J., Kammenga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans.">Genome-Wide Gene Expression Analysis in Response to Organophosphorous Pesticide Chlorpyrifos and Diazinon in C. elegans.</a> PloS one. 5, 1-8 (2010).</li><li>Niwa, R., Hada, K., Moliyama, K., Ohniwa, R., Tan, Y., Olsson-Carter, K., Chi, W., Reinke, V., Slack, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor.">elegans sym-1 is downstream target of the hunchback-like-1 developmental timing transcription factor.</a> Cell Cycle. 8, 4147-4154 (2009).</li><li>Kershner, A., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF,+a+conserved+stem+cell+regulator.">Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.</a> PNAS. 107, 3963-3941 (2010).</li><li>Hammock, E., Eagleson, K., Barlow, S., Earls, L., Miller, D., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain.">Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain.</a> Neural Development. 5, 1-14 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo studio abbiamo sviluppato un semplice protocollo facile da usare che può essere usato per determinare profili di espressione genica di base vari fenomeni biologici. In questo protocollo, l&#39;RNA totale è stato isolato e trattato con DNasi I. Il nostro metodo di isolamento dell&#39;RNA è stata notevolmente semplificata dall&#39;eliminazione fenolo e l&#39;utilizzo di resine affinità per ripulire 5,6. In breve, C. elegans cuticule membrane cellulari e sono state incrinate, aperto da nematodi congelamento con azoto liquido e rettifica con resina molecolare. Successivamente, l&#39;RNA estratto è stato trattato con DNasi I prima sintesi del DNA. Il passaggio successivo è stato aggiunto a minimizzare ibridazioni aspecifiche; campioni di RNA contaminato da DNA genomico potrebbe introdurre interferenze e producono molti falsi positivi. Poi, tipo selvatico e VSM-1 (ok1468) cDNA mutanti sono stati taggati con Cy3 e Cy5 sequenze di acquisizione e ibridato su C. microarray elegans ottenuti tramite il programma GCAT. Questo sistema è più sensibile rispetto a prodotti simili, e il suo sofisticato disegno bicolore diminuisce il numero di array necessari, con conseguente maggiore efficienza dei costi di tempo e 7,8. Inoltre, questo sistema non richiede attrezzature specializzate di altri sistemi simili, quindi, questa procedura potrebbe essere realizzato in qualsiasi ambiente standard di laboratorio 7. In terzo luogo, le immagini fluorescenti serie ibridata sono stati analizzati utilizzando il software a sorgente aperto MAGICTool, e profili di espressione genica sono stati creati. MAGICTool è un programma di facile uso che può essere facilmente utilizzato da individui di ogni livello di esperienza, dalla laurea al post-dottorato. Tuttavia, la performance ottimale richiede grande disponibilità di memoria. Infine, geni candidati ottenuti con l&#39;analisi microarray sono state validate con PCR in tempo reale. Anche se l&#39;approccio genomico è un metodo efficace per studiare fenomeni biologici, ci sono alcune limitazioni si dovrebbe prendere in considerazione. In primo luogo, nonostante la specificità di questo protocollo microarray, trascrizioni all&#39;interno di una famiglia sono indistinguibili l&#39;uno dall&#39;altro se il oligonucleotide stampata è tratto da sequenze conservate. PCR in tempo reale compensa somiglianze all&#39;interno di una famiglia di geni e può anche distinguere tra isoforme specifiche dello stesso gene. Tuttavia, con PCR in tempo reale è una sfida per trovare controlli vero che sono espressi allo stesso modo per tutta la durata della vita di un organismo. A causa di questo, i risultati saranno relativi. Un approccio proteomico è una alternativa alla nostra tecnica. Singole proteine ​​possono essere isolate mediante gel elettroforesi 2D e identificati con la spettrometria di massa. I nostri studi mostrano in particolare che molti membri delle principali proteine ​​dello sperma (MSP) famiglia sono indotti a VSM-1 nematodi mutante con una maggiore densità sinaptica. MSP sono unici a C. elegans e sono responsabili della maturazione dell&#39;ovocita e l&#39;ovulazione. Chai 1 ha dimostrato che la regolamentazione del numero Bouton presinaptica e la dimensione a livello della giunzione neuromuscolare nei moscerini della frutta è probabile controllato da molecole contenenti principali domini delle proteine ​​dello sperma. Studi di Tsuda e colleghi hanno dimostrato due principali domini di proteine ​​dello sperma possono servire come leganti per i recettori Ephrine, innescando cascate della tirosin-chinasi del recettore di segnalazione. Inoltre, l&#39;analisi real time PCR convalidato e ristretto i risultati dei microarray a un membro della famiglia msp, msp-32. Nel loro insieme, il lavoro qui presentato rappresenta un genoma analisi di un dilemma centrale neuroscienze così come la potenza di ricerca universitari nello sforzo scientifico.

<table border="1"><tbody><tr><td> Componente </td><td> Catalogo </td><td> Azienda </td></tr><tr><td> Più tardi dell&#39;RNA </td><td> AM7020 </td><td> Ambion </td></tr><tr><td> Molecolare resina Rettifica </td><td> 786-138 </td><td> Gbiosciences </td></tr><tr><td> RNeasy Mini Kit </td><td> 74104 </td><td> Qiagen </td></tr><tr><td> RNasi-free Agarose LE </td><td> AM9040 </td><td> Ambion </td></tr><tr><td> NorthernMax Gly 10X Gel Prep / Running Buffer </td><td> 8678 </td><td> Ambion </td></tr><tr><td> RNA Millennium Marker </td><td> AM7150 </td><td> Ambion </td></tr><tr><td> Gliossale campione colorante di caricamento </td><td> 8551 </td><td> Ambion </td></tr><tr><td> DNasi set </td><td> 79254 </td><td> Qiagen </td></tr><tr><td> RNeasy MinElute Kit </td><td> 74204 </td><td> Qiagen </td></tr><tr><td> RT enzimi e tampone di reazione 5X </td><td> RT300320 </td><td> Genisphere </td></tr><tr><td> Array 350 </td><td> W300180 </td><td> Genisphere </td></tr><tr><td> QIAquick PCR Purification Kit </td><td> 28104 </td><td> Qiagen </td></tr><tr><td> SSC 20X </td><td> 82021-4846 </td><td> VWR </td></tr><tr><td> Tranciati Salmone DNA dello sperma </td><td> AM9680 </td><td> Ambion </td></tr><tr><td> SDS Soluzione </td><td> V6551 </td><td> Promega </td></tr><tr><td> DTT EM </td><td> 3860 </td><td> VWR </td></tr><tr><td> iScript Kit di sintesi del DNA </td><td> 170-8890 </td><td> BioRad </td></tr><tr><td> iQ SYBR Green Supermix </td><td> 170-8880 </td><td> BioRad </td></tr></tbody></table>

determining genetic expression profiles in c. elegans using microarray and real-time pcr

Sinapsi sono composti da una zona presinaptica attivo nella segnalazione cellulare e un terminale postsinaptici nella cella di destinazione. Nel caso di sinapsi chimiche, i messaggi vengono trasportati da neurotrasmettitori rilasciati dai terminali presinaptici e ricevuti dai recettori sulle cellule postsinaptici. Le nostre ricerche precedenti in Caenorhabditis elegans ha dimostrato che VSM-1 regola negativamente esocitosi. Inoltre, l&#39;analisi di sinapsi in VSM-1 mutanti hanno mostrato che gli animali privi di una connettività completamente funzionale VSM-1 sono aumentati sinaptica. Sulla base di questi risultati preliminari, abbiamo ipotizzato che C. elegans VSM-1 può giocare un ruolo cruciale nella sinaptogenesi. Per verificare questa ipotesi, a doppio etichettati analisi microarray è stata eseguita, e profili di espressione genica sono stati determinati. In primo luogo, l&#39;RNA totale è stato isolato, inversamente trascritto in cDNA, e ibridato al DNA microarray. Poi, in silico-analisi di ibridazione sonda fluorescente rivelato induzione significativo di molti geni che codificano per i membri della famiglia principali proteine ​​dello sperma (MSP) in mutanti con sinaptogenesi migliorata. MSP è il principale componente di sperma in C. elegans e sembrano segnale maturazione degli ovociti nematodi e l&#39;ovulazione. Nel moscerino della frutta, Chai e colleghi hanno dimostrato che 1 MSP-come le molecole regolano il numero e la dimensione bouton presinaptica a livello della giunzione neuromuscolare. Inoltre, l&#39;analisi eseguita da Tsuda e collaboratori due suggerito che MSP può agire come leganti per i recettori Ef e innescare tirosin-chinasi del recettore cascate di segnalazione. Infine, l&#39;analisi PCR in tempo reale corroborata che il gene che codifica per MSP-32 è indotta a VSM-1 (ok1468) mutanti. Nel loro insieme, la ricerca effettuata dal nostro laboratorio ha dimostrato che VSM-1 mutanti hanno un significativo aumento della densità sinaptica, che potrebbe essere mediata da MSP-32 di segnalazione.

Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Analisi microarray è stato condotto per determinare profili di espressione genetica<em&gt; C. elegans</em&gt;, E real-time PCR è stato utilizzato per convalidare e quantificare i dati di microarray.

Determinare profili di espressione genetica in C. elegans Utilizzo Microarray e Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

determining-genetic-expression-profiles-c-elegans-using-microarray

Research

JoVE Journal

Biology

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

22.9K Views.  Southwestern Oklahoma State University. Analisi microarray è stato condotto per determinare profili di espressione genetica C. elegans, E real-time PCR è stato utilizzato per convalidare e quantificare i dati di microarray.

Video: Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Ricerca

Biologia

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Analisi di espressione genica globale Utilizzando un Zebrafish piattaforma Microarray oligonucleotidi

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

Quantitativa Real-Time PCR utilizzando il Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Analisi dell&#39;espressione genica in Borer Emerald Ash ( Agrilus planipennis) Utilizzando quantitativa Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Profilatura di pre-micro-RNA e microRNA con Quantitative Real-time PCR (qPCR) Array

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimica e High Throughput valutazione microscopica di massa grassa in<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Antibody colorazione in<em&gt; C. Elegans</em&gt; Uso di &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

Linear Amplificazione Mediata PCR - Localizzazione di Genetica Elementi e caratterizzazione di Unknown Aggirare DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

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.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Base-Probe Real-time PCR Approcci per la misurazione quantitativa dei microRNA

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...