Questo lavoro è stato finanziato da una borsa di studio della National Science Foundation (codice di autorizzazione MCB-0447750).

<ol>
	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

Nessun conflitto di interessi dichiarati.

La calotta di ghiaccio metodo presentato qui uno scienziato permette di raccogliere campioni di tessuto da centinaia o addirittura migliaia di singole piante in un solo giorno ed efficiente estrarre il DNA genomico da questi campioni per l&#39;utilizzo in reazioni genotipizzazione. Dando uno scienziato individuo la possibilità di genotipo migliaia di piantine in un breve periodo di tempo, la calotta di ghiaccio metodo ha il potenziale di facilitare una serie di esperimenti genetici che altrimenti non sarebbero pratici da eseguire. Numerosi esempi sono forniti di seguito. <ol><li> Mappatura genetica. Scala fine mappatura genetica può essere accelerato se il ricercatore è in grado di identificare eventi di ricombinazione all&#39;interno di uno specifico intervallo lungo del cromosoma 3. Se questo intervallo comporta una distanza molto breve genetica, poi gli eventi di ricombinazione sarà relativamente rara nella popolazione mappatura segreganti. Per questo motivo, può essere necessario a migliaia schermo delle singole piante per trovare il re desideratoindividui ricombinanti. Questo processo potrebbe essere notevolmente accelerato mediante l&#39;utilizzo di Ice-Cap. Anche nell&#39;era del genoma ri-sequenziamento, è ancora necessario dimostrare direttamente che una mutazione è direttamente responsabile di un fenotipo di interesse. In questi casi, sarà necessario separare la mutazione di interesse lontano da altre mutazioni legate strettamente al fine di dimostrare una mutazione candidato è in realtà causale. In queste situazioni, Ice-Cap fornirebbe un metodo efficace per rompere il collegamento tra una coppia di mutazioni strettamente collegati. </li><li> Multi-mutanti. E &#39;diventato comune per i ricercatori che studiano Arabidopsis di combinare alleli mutanti di diversi loci distinti in un singolo individuo al fine di superare la ridondanza funzionale 4-6. Come esempio, consideriamo un caso in cui si vuole combinare alleli mutanti da quattro loci differenti in un singolo impianto attraverso incrocio genetico. Per questo progetto, sarà necessario generare unn individuo che è eterozigote in questi quattro loci come parte del sistema di attraversamento. Quando questo quadruplo eterozigote semi-set, il quadruplo omozigote dovrebbe essere presente al ritmo di uno su 256 nella popolazione risultante della progenie. Lo screening per questa persona rara con i metodi tradizionali sarebbe abbastanza laborioso. In confronto, con Ice-Cap per questo progetto avrebbe fornito lo scienziato con una strategia efficace per identificare i omozigote rara quadrupla in una sola generazione. </li><li> iTILLING. Il nostro laboratorio ha recentemente descritto un nuovo approccio allo screening per la presenza di mutazioni indotte nei geni di interesse in Arabidopsis 7. Questo metodo è una variante di una tecnica consolidata chiamata TILLING che viene utilizzato per trovare mutazioni nella popolazione ordinata di individui trattati con un mutageno chimico 8-10. Abbiamo chiamato il nostro metodo iTILLING perché è una versione personalizzata di TILLING che permette ad un individual scienziato a realizzare qualcosa che ha sempre richiesto un grande team di ricerca da completare. Ice-Cap costituisce parte integrante del metodo iTILLING 7, come descritto di seguito. </li></ol> L&#39;idea alla base iTILLING è quello di produrre una popolazione mutante effimero, che è in contrasto con le popolazioni resistenti mutante creato per i tradizionali progetti TILLING. Per iTILLING, i semi di una popolazione gonfiato M2 vengono seminati in blocchi di ghiaccio dal 50-100 Cap, e il DNA viene estratto utilizzando il ghiaccio-Cap metodo. Piantine di Arabidopsis vengono poi poste a 4 ° C mentre screening delle mutazioni viene eseguita utilizzando il ghiaccio-Cap DNA prep. Piantine di pomodoro sono conservati a 12 ° C.. Una volta piantine mutazioni portando desiderati sono stati identificati, sono recuperati dalla Ice-Cap piastre e trasferiti a terra. iTILLING non è destinato a fornire una lunga popolazione mutante per una comunità intera ricerca a schermo. Invece, iTILLING fornisce una strategia con la qualeuno scienziato può schermo una popolazione mutante su misura per le mutazioni in una manciata di geni in modo rapido, efficiente e ad un costo relativamente basso. Abbiamo trovato Ice-Cap di essere un metodo efficace per la coltivazione e la genotipizzazione Arabidopsis, il pomodoro e riso. Ci aspettiamo che le altre specie di piante devono essere suscettibili di Ice-Cap. Una limitazione sulla varietà di specie che possono essere ospitati da Ice-Cap è la dimensione dei semi. Solo le piante con semi abbastanza piccolo da entrare nei pozzetti di una piastra a 96 pozzetti sarà compatibile con calotta di ghiaccio nella sua attuale configurazione.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, NJ </td><td> 2000 Geno / Grinder </td><td> Macchina per scuotimento piastre Root per l&#39;estrazione del DNA </td></tr><tr><td> ABgene Thermo-Sealer </td><td> ABgene USA </td><td> AB-0384 </td><td> Manuale di tenuta della macchina di calore utilizzato per lastre di root prima di disgregazione del tessuto </td></tr><tr><td> Centrifuga dotata di supporti per micropiastre oscillante </td><td> Beckman Coulter </td><td> Allegra 25R </td><td> Qualsiasi centrifuga con supporti per micropiastre oscillante sarebbe adatta </td></tr><tr><td> Microfill Dispenser per micropiastre </td><td> Biotek www.biotek.com </td><td> AF1000A </td><td> Dispenser automatico di liquido per micropiastre riempimento </td></tr><tr><td> Bialetti marchio17x11 pollici Foglio commerciale Cookie Grandi </td><td> Obiettivo Department Store </td><td> www.bialetti.com </td><td> Questo strato di biscotto specifico è la profondità ideale per Ice-Cap </td></tr><tr><td> Custom Made Rack in metallo </td><td> Ferramenta </td><td></td><td> Cfr. il testo per la descrizione completa </td></tr><tr><td> 33 litri in plastica contenitore </td><td> Obiettivo Department Store </td><td> Sterilite Marca </td><td> Per Ice-Cap Fontana. Stoccaggio scatola di plastica abbastanza grande da contenere il foglio 17x11 cm cookies </td></tr><tr><td> 30 gallone Pompa Fontana ore </td><td> Sunterra </td><td> 104506 </td><td> Piccolo sommergibile pompa fontana disponibile nei negozi di giardino </td></tr><tr><td> Tubo per pompa fontana </td><td> Ogni laboratorio società di fornitura </td><td></td><td> Tubo compatibile con pompa fontana </td></tr><tr><td> Pinza in plastica </td><td> Ferramenta </td><td></td><td> Fascetta per tenere tubo cookiesfoglio </td></tr><tr><td> Coperchi di plastica trasparente dal basso Falcon Microtest piatto piastre a 96 pozzetti in polistirene </td><td> BD Falcon www.bdbiosciences.com </td><td> 351172 </td><td> Chiaro coperchi di plastica per la copertura di ghiaccio-Cap Piastre Seedling </td></tr><tr><td> 3M Micropore nastro chirurgico </td><td> Fisher Scientific </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch diametro sfere in acciaio inox </td><td> Tecnologie Hartford, Rocky Hill, CT </td><td> 034-006-1K </td><td> Utilizzato per distruggere il tessuto radice dopo la raccolta </td></tr><tr><td> 3 bande pollici capelli elastici </td><td> Drogheria locale </td><td></td><td> Utilizzati per fissare Piastra Root to Plate Seedling </td></tr><tr><td> 4 spiedini di legno mm di diametro </td><td> Drogheria locale </td><td></td><td> Questi spiedini sono tipicamente utilizzati per la preparazione kabobs </td></tr><tr><td> Fisher Scientific Nunc marchio, 1-mL piastre filtranti senza fritta </td><td> Fisher Scientific </td><td> 278012 </td><td> 96-pozzetti Seedling con fori nella parte inferiore. </td></tr><tr><td> Fisherbrand 96-Well Tall-camino Piastre PCR </td><td> Fisher Scientific </td><td> 14-230-242 </td><td> 96-Root e Piastre </td></tr><tr><td> Agar - colture cellulari testati </td><td> Sigma-Aldrich </td><td> A1296 </td><td></td></tr><tr><td> 4-Morpholinoethanesulfonic acido (MES) </td><td> Sigma-Aldrich </td><td> M2933 </td><td></td></tr><tr><td> Murashige e Skoog (MS), miscela di sale basale </td><td> Sigma-Aldrich </td><td> M-5524 </td><td></td></tr><tr><td> Peel calore facile tenuta foglio </td><td> ABgene USA </td><td> AB-0745 </td><td> Calore pellicola sigillante per sigillare piastre radice prima di disgregazione del tessuto </td></tr><tr><td> Anatra Marca extra largo nastro di imballaggio, 3,00 &quot;x 54,6 m </td><td> OfficeMax www.officemax.com </td><td> Articolo no. 21242139 </td><td> Nastro adesivoper micropiastre sigillatura contenenti estratti di DNA per la conservazione </td></tr><tr><td> Tenuta Roller </td><td> Bio-Rad </td><td> MSR-0001 </td><td> Mano lo strumento per lo stampaggio a rotazione nastro adesivo di tenuta su micropiastre </td></tr><tr><td> 96-blocco di metallo e termica </td><td> Cole-Parmer </td><td> 36400-66 </td><td> Utilizzato per congelare le tavole Root per la raccolta dei tessuti </td></tr><tr><td> Carta da filtro Whatman </td><td> Whatman internazionale </td><td> 1002 090 </td><td> Utilizzato per le sementi superficie sterilizzazione </td></tr><tr><td> Pipetta Pasteur monouso </td><td> Fisher Scientific </td><td> 13-678-6A </td><td> 5 ¾ &quot;usa e getta pipetta Pasteur utilizzato per trasferire i semi per piastra Seedling </td></tr><tr><td> Pyrex teglia (20 cm x 30 cm) </td><td> Obiettivo Department Store </td><td></td><td> Usato per costruire una ghiaccio secco / etanolo da bagno. </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

Sta diventando comune per gli scienziati impianto di sviluppare progetti che richiedono la genotipizzazione di un gran numero di piante. Il primo passo in qualsiasi progetto di genotipizzazione è quello di raccogliere un campione di tessuto da ogni singolo impianto. L&#39;approccio tradizionale a questo compito è quello di piante campione one-at-a-tempo. Se si vuole genotipo centinaia o migliaia di individui, tuttavia, usando questa strategia i risultati in un collo di bottiglia significativo in cantiere genotipizzazione. La calotta di ghiaccio metodo che descriviamo qui fornisce un elevato throughput soluzione a questa sfida, consentendo uno scienziato per la raccolta di tessuto da diverse migliaia di piantine in un solo giorno 1,2. Questo livello di velocità è reso possibile dal fatto che il tessuto viene raccolto da piante 96-at-a-tempo, piuttosto che un at-a-tempo. La calotta di ghiaccio metodo fornisce una piattaforma integrata per l&#39;esecuzione di crescita delle piantine, raccolta dei tessuti, e l&#39;estrazione del DNA. La base per Ice-Cap è la crescita di seedliNGS in un paio sovrapposti di piastre a 96 pozzetti. I pozzetti della piastra superiore contengono spine di media crescita agar su cui semenzali singoli germinare. Le radici crescono verso il basso attraverso i media agar, uscire la piastra superiore attraverso un foro, e passare in acqua piastra inferiore che contiene. Per la raccolta di tessuto per l&#39;estrazione del DNA, l&#39;acqua nella piastra inferiore contenente tessuto radice è rapidamente congelato, mentre le piantine nella piastra superiore rimanere a temperatura ambiente. La piastra superiore è poi staccato dalla piastra inferiore, si possa ottenere una piastra con 96 campioni di radice di tessuto congelato nel ghiaccio e un piatto con 96 piantine vitali. La tecnica si chiama &quot;Ice-Cap&quot; perché utilizza il ghiaccio per catturare il tessuto radice. La piastra a 96 pozzetti contenenti le piantine possono poi avvolto in un foglio e trasferiti a bassa temperatura. Questo processo sospende l&#39;ulteriore crescita delle piantine, ma non influenza la loro vitalità. Una volta che l&#39;analisi del genotipo è stata completata, piantine con il genotipo desiderato può essere trasferita dal 96-bene pin ritardo a terra per ulteriore moltiplicazione. Abbiamo dimostrato l&#39;utilità del ghiaccio-Cap metodo utilizzando Arabidopsis thaliana, pomodoro, e le piantine di riso. Ci aspettiamo che il metodo deve essere applicabile anche ad altre specie di piante con semi abbastanza piccolo da entrare nei pozzetti di piastre a 96 pozzetti.

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping | Biology | Video

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

La calotta di ghiaccio metodo permette di coltivare le piante in piastre con 96 pozzetti e non distruttivo dei tessuti radice raccolta da ogni piantina. DNA estratto da questo tessuto radice può essere utilizzata per le reazioni di genotipizzazione. Abbiamo scoperto che Ice-Cap funziona bene per<em&gt; Arabidopsis thaliana</em&gt;, Pomodoro, e piantine di riso.

Ice-Cap: Un metodo per la Crescita Arabidopsis E piante di pomodoro in piastre con 96 pozzetti di High-Throughput Genotipizzazione

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

ice-cap-method-for-growing-arabidopsis-tomato-plants-96-well-plates

Research

Education

JoVE Journal

JoVE Core

Biology

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Plant Biology

Plant Reproduction

SCIENTISTS IN ACTION

22.7K Views.  University of Wisconsin-Madison. La calotta di ghiaccio metodo permette di coltivare le piante in piastre con 96 pozzetti e non distruttivo dei tessuti radice raccolta da ogni piantina. DNA estratto da questo tessuto radice può essere utilizzata per le reazioni di genotipizzazione. Abbiamo scoperto che Ice-Cap funziona bene per Arabidopsis thaliana, Pomodoro, e piantine di riso.

Video: Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Ricerca

Biologia

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Un rapido ad alta velocità Metodo per ribonucleoproteine ​​Mapping (RNP) su Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Misura Caenorhabditis elegans Life Span in 96 piastre per microtitolazione Bene

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un metodo ad alta velocità per la misura della velocità di filtrazione glomerulare in topi coscienti

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Misurazione spaziale e temporale Ca<sup&gt; 2 +</sup&gt; Segnali in Arabidopsis Piante

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Misura Stress ossidativo Resistenza<em&gt; Caenorhabditis elegans</em&gt; In 96 pozzetti micropiastre

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

High-throughput CRISPR vettore Costruzione e caratterizzazione di modifiche del DNA da Generazione di Roots pomodoro Peloso

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...