We thank Stephen Blazie, Karen Anderson, Josh LaBaer for advice and discussion. Karen Anderson, John Chaput, and Josh LaBaer for sharing reagents and instrumentation, Michael Gaskin and Andrea Throop for technical advise and protocols. Justin Wolter is a Maher scholar and thanks the Maher family for their generous support.

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This work is supported by funds from the College of Liberal Arts and Science and the Biodesign Institute at Arizona State University and NIH Exploratory/Developmental Research Grant 1R21CA179144-01A1.

Assay 3&#39;LIFE מזהה מטרות מירנה תפקודיות ב3&#39;UTRs בתפוקה גבוהה. assay זה שימושי לחוקרים המבקשים לזהות באופן ניסיוני במספר רב של מטרות המשוערות למירנה עניין שלהם. Assay 3&#39;LIFE הוא גישה רבת עוצמה לשאילתא לרגולציה מונעת 3&#39;UTR, שבassay מספק מדד תפקודי של מיקוד מירנה, והבדיקות בינארי של ?...

המטרה הכוללת של שיטה זו היא לזהות ולמפות בדיוק microRNA מטרות (מירנה) בתפוקה גבוהה. MiRNAs הוא RNAs קידוד שאינו אנדוגני ~ 22 נוקלאוטידים באורך. בעקבות שעתוק ועיבוד, miRNAs הבוגר משולבים בקומפלקס חלבון הנקרא RNA מושרה השתקה מורכב (RISC). כל מירנה מנחה את RISC למקד אלמנטים הממוקמים בעיקר באזורי 3&#39;untranslated (3&#39;UTRs) של RNAs שליח (mRNAs), וכתוצאה מכך גם דיכוי תרגום או מחשוף mRNA 1. מירנה להכיר אתרי יעד המבוססים על ווטסון-קריק הסטנדרטי וG: U לנענע זיווג בסיס, והם מנוונים בטבע, המכיל זוגות בסיסים תואמים מרובים ובלטה אזורים. miRNAs רבים נשמר באופן רחב מצמחים לבני האדם 2,3, שבו הם משחקים במגוון רחב של תפקידים ביולוגיים. בmetazoans miRNAs יכול להשפיע על תהליכים ביולוגיים רבים, כולל החלטות גורל תא 4, עיתוי התפתחותי 5 </sup&gt;, ולעתים קרובות מפגין דפוסי ביטוי רקמות ספציפיים 6,7. misexpression מירנה יכול גם לגרום לויסות הגנים חריגה, אשר יכולה להיות השפעה מהותית על מבוססת אך ורק על התפקוד של גני מטרת התנהגות תא. ככזה, miRNAs צמוד למגוון רחב של מחלות, כולל ניוון מוחיים 8,9, סוכרת וסרטן 10 11. Bioinformatic וגישות רטוב ספסל מצביעים על כך שכל מירנה עשויה להיות מסוגל מיקוד מאות עד אלפי mRNAs הברור 12-14, מצביע על כך שתפוקה גבוהה או הגנום גישות רחבות נדרשות לחקור בריכה גדולה זו של אינטראקציות פוטנציאליות. זיהוי גני המטרה הוא מרכיב קריטי בתפקוד מירנה מכניסטי הגדרה, וכדי לעשות זאת חוקרים חייבים להיות מסוגלים לחשוף מטרות בקנה מידה גדולה. כמה גישות פותחו כדי לזהות מטרות מירנה, כולל אלגוריתמי תחזית bioinformatic, רצף תפוקה גבוההשל ממוקד mRNAs, וכתב מבחני מבוסס. כל הגישות הללו עוצמות וחולשות הגלומות. בהתחשב בכך שמיקוד מירנה מונחה על ידי סגוליות רצף, בעיקר של נוקלאוטידים 2-6 של מירנה (שמכונה אזור הזרע), כמה אלגוריתמים פותחו לחזות מטרות מירנה לאורך הגנום של אורגניזמים רבים. אלגוריתמים אלה מאומנים באמצעות המוטיבים בסיס שיוך הנצפים של מטרות מירנה תוקף, ולעתים קרובות לנצל פרמטרים כגון זיווג מחמיר זרע, שימור אתר, ו / או יציבות התרמודינמית 15. בעוד מסננים אלה לחדד מספר רב של מטרות המשוערות עם השלמה מספיק למטרות ביטחון בלבד גבוהות, הם עלולים שלא לכלול מיני אתרים ספציפיים ואינם קנוני מירנה יעד, שהראיות האחרונות עולה נפוצות 16-24. יתר על כן, תחזיות אלו אינן לוקחות בחשבון את מנגנוני עיבוד mRNA שלא לכלול אתרי יעד מירנה, כגון polyadenylation החלופי25, עריכת RNA 26, מתילציה RNA 27, ומחייב שיתוף פעולה. ככזה, שיעורים חיוביים ושקר שקר שליליים גבוהים דווחו לאלגוריתמים רבים 22,24,28. בעוד אלגוריתמים אלה הם שימושיים כדי לזהות מטרות מירנה מועמד לאימות ניסיוני שלאחר מכן, שיעורי שגיאה הגבוהים אלה מגבילים את היעילות של גישות bioinformatic לאיתור יעד מירנה שיטתית. לחקור באופן שיטתי לאינטראקציות בין מירנה נתון ו3&#39;UTRs פוטנציאלי הממוקד פיתחנו assay תפוקה גבוהה נקרא פלורסנט זיהוי של אלמנטים פונקציונליים ב3&#39;UTRs (3&#39;LIFE) 24. צעדים זה assay אינטראקציות ישירות ודיכוי translational של 3&#39;UTR הבדיקה על ידי שאילתא מירנה באמצעות מערכת כתב בלוציפראז כפולה. במערכת זו, 3&#39;UTR של גן של עניין הוא משובט במורד הזרם של מסגרת קריאת גחלילית בלוציפראז (fluc) כתב. חסרונות הכתבtruct הוא cotransfected עם שאילתא מירנה בתאי HEK293T. מיקוד מירנה נקבע על ידי מדידת השינוי ביחס בין fluc המבחן :: כתב 3&#39;UTR וכתב בלוציפראז Renilla אינו ספציפי שני. חשוב לציין, מבחני בלוציפראז לזהות אינטראקציות מירנה / mRNA תפקודיים המשפיעות על תפוקת translational של הכתב. זהו יתרון מרכזי על פני שיטות מסורתיות כדי לזהות רגולציה מירנה, כגון RT-qPCR וכתמים מערביים, שבזו עוקפת הבדלים בשפלת mRNA ודיכוי translational, כמו גם שינויים בשפע חלבון עצמאי של רגולציה מבוססת 3&#39;UTR. מבחני בלוציפראז משמשים באופן נרחב כדי לאמת מטרות מירנה ישירות בגלל פשטותם היחסית וברגישות, עדיין השימוש בם במסכי תפוקה גבוהה הוא מוגבל על ידי עלויות גבוהות כרוכות בחומרים כימיים מתכלים, חוסר ספריות 3&#39;UTR ממקורות ציבוריים, והעדר של prot לוציפראז הסטנדרטיocols, שהוביל לקשיים בהשוואת דיכוי פונקציונלי על פני מערכי נתונים מרובים. כדי להקל על השימוש של assay 3&#39;LIFE, יש לנו דגש על פישוט של עיצוב ניסיוני, שימוש בחומרים כימי transfection 24 ולוציפראז לא מסחרי 29, יצירת ספריית 3&#39;UTR המתעדכנת באופן קבוע ומורחבת, והוא זמין ב מאגר פלסמיד ציבור 30. יכולת הרחבה של assay 3&#39;LIFE מאפשרת הקרנה של ספריית 3&#39;UTR גדולה למיקוד על ידי מירנה ניתנת ללא הטיית המסך לכיוון הגנים שזוהו bioinformatically. בנוסף לבדיקת אינטראקציות הקנונית וחזו, גישה שיטתית זו מאפשרת זיהוי מטרות רומן מונעות באמצעות אינטראקציות הלא-הקנונים ו / או מינים ספציפיות. חשוב לציין, את ההשפעה של מירנה מיקוד על ייצור חלבון הוא הבין בדרך כלל לגרום לדיכוי translational הצנוע 15,31 &lt;/ Sup&gt;, המצביע על כך תפקיד עיקרי של הרגולציה מירנה הוא לכוונן תפוקת חלבון, להגן מפני רמות חריגות של ביטוי גנים, ומספק חוסן לתא תוכניות ספציפיות 32,33. הרגישות של assay בלוציפראז בשילוב עם המספר הגדול מיסודו של אינטראקציות מירנה / mRNA שליליים במסך 3&#39;LIFE מאפשרת זיהוי של השפעות עדינות של מירנה מיקוד על מספר גדול של גנים, וזיהוי של מרכיבים רבים של רשתות גנים ש מוסדרים על ידי מירנה נתנה 24. כאן אנו מתארים את פרוטוקול 3&#39;LIFE, ולהפגין זה היתכנות על ידי הקרנת שני miRNAs המאופיין היטב, miR-10b ולתת-7C נגד פנל של 275 3&#39;UTRs האנושי (איור 1).

<table border="0" cellpadding="0" cellspacing="0" width="406">
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			<td height="21" style="height:21px;width:216px;">Reagents</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well Shuttle System</td>
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	</tbody>
</table>

detection of mirna targets in high-throughput using the 3'life assay

פלורסנט זיהוי של אלמנטים פונקציונליים ב3&#39;UTRs (3&#39;LIFE) מאפשר זיהוי המהיר של מטרות של miRNAs הספציפי בתוך מערך של מאות 3&#39;UTRs שאילתא. זיהוי יעד מבוסס על assay הכפול לוציפראז, אשר מזהה מחייב ברמת ה- mRNA על ידי מדידת תפוקת translational, נותנת קריאת נתונים פונקציונליות של מירנה מיקוד. 3&#39;LIFE משתמש מאגרים שאינן קניינית וריאגנטים, וספריות כתב זמינות בפומבי, מה שהופך את מסכי הגנום אפשרי וחסכוני. ניתן לבצע 3&#39;LIFE או במעבדה הגדרה סטנדרטית או לשנותם באמצעות רובוטים טיפול נוזליים ומכשור תפוקה גבוהה אחר. אנו מדגימים את הגישה באמצעות בסיס הנתונים של 3&#39;UTRs האנושי משובט ב96-גם צלחות, ושני miRNAs בדיקה, לתת-7C וmiR-10b. אנו מדגימים כיצד לבצע הכנת DNA, transfection, תרבות תא ומבחני בלוציפראז בפורמט 96-היטב, ולספק כלים לנתוניםניתוח. לסיכום 3&#39;LIFE הוא מאוד לשחזור, מהיר, שיטתי, ומזהה מטרות ביטחון גבוהות.

קובץ פלט luminometer מכיל מדידות גלם לשני חלבוני לוציפראז הגחלילית וRenilla. פורמט גלם זה תואם עם &quot;3&#39;LIFE - ניתוח צלחת אחת&quot; ו- &quot;3&#39;LIFE - ניתוח multiplate&quot; גיליונות אלקטרוניים זמין מאתר האינטרנט של המעבדה Mangone <a href="http://www.mangonelab.com">(www.mangonelab.com).</a> הגיליון האלקטרוני ניתוח צלח...

Watch this Scientific Journal Video about Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay at JoVE.com

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

זיהוי של מטרות מירנה בתפוקה גבוהה באמצעות assay 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

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JoVE Journal

Biology

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

10.0K Views.  Arizona State University. Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Video: Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

תפוקה גבוהה Saccharification Assay חומרים Lignocellulosic

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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&#900;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%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

הגנום כולו מסך עבור מטרות מירנה שימוש יעד המשימה ספריית מזהה

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

תפוקה גבוהה התגבשות של חלבונים בממברנה שימוש בשיטה Lipidic Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

תפוקה גבוהה מיפוי פיזי של הכרומוזומים באמצעות אוטומטי<em&gt; באתרו</em&gt; הכלאה

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

מחיקות Gene הגנום ב<em&gt; סטרפטוקוקוס sanguinis</em&gt; על ידי תפוקה הגבוהה PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

חיזוי מבנה המשני RNA באמצעות SHAPE תפוקה גבוהה

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

הקרנה פונקציונלית תפוקה גבוהה באמצעות assay בלוציפראז Dual-זוהר תוצרת בית

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

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

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

תפוקה גבוהה<em&gt; Danio rerio</em&gt; Assay הוצאה אנרגיה

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

שיטה סטנדרטית עבור תפוקה גבוהה עיקור של תודרנית זרעים

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...