אנו מבקשים להודות לחברי הצוות לאם וואן BAC מעבדה מערך במיוחד Miwa סוזוקי בריאן צ&#39;י להכנת מאמר זה. עבודה זו נתמכה על ידי קרנות מן המכונים הקנדי לבריאות מחקר הגנום קנדה / הגנום קולומביה הבריטית, ו-NIH / NIDCR מענק RO1 DE15965-01.

בדיקה סימון הערה: להגביל את החשיפה של צבעים Cye לאור בכל הזמנים (זו יכולה להיות מושגת על ידי עבודה באזור חשוך או על ידי מיגון צינורות עם כיסוי כמו רדיד אלומיניום) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שלב: (Setup 1 שפופרת תגובה עבור התייחסות 1 שפופרת התגובה למדגם) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> DNA (25-400 נ&quot;ג) </li><li style=";text-align:right;direction:rtl"> 5 μL של חיץ primers 5X אקראית (ריכוז סופי: 5X Promega Klenow חוצץ ו 7 מיקרוגרם / octamers אקראי μL) </li><li style=";text-align:right;direction:rtl"> מדולל נפח 17.0 הכולל μL מזוקקים עם H 2 O </li></ul></li><li style=";text-align:right;direction:rtl"> מרתיחים במשך 10 דקות ב 100 ° C. העברה מיידית קרח למשך דקה 1. </li><li style=";text-align:right;direction:rtl"> הוסף 4 μL תמהיל dNTP 10X (2mm dATP, dGTP, dTTP, dCTP 1.2 מ&quot;מ). </li><li style=";text-align:right;direction:rtl"> הוסף CyeDyes: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף μL 2 (2 nmoles) של dCTP סיי-3 שכותרתו ל-DNA Sample </li><li style=";text-align:right;direction:rtl"> הוסף μL 2 (2 nmoles) של סיי-5 dCTP שכותרתו הפניה DNA (למשל, ה-DNA הגנומי Novagen אדם) </li></ul></li><li style=";text-align:right;direction:rtl"> הוסף 2.5 μL של Klenow (9 U / μL, Promega) ומערבבים. </li><li style=";text-align:right;direction:rtl"> לדגור על 37 מעלות צלזיוס למשך הלילה * (~ 18 שעות). * הפעם הכלאה בין לילה יכול להיות מותאם בין 14 ל -24 שעות מבלי להשפיע לרעה על תהליך תיוג כדי להתאים ללוח הזמנים מעבדה. </li></ol> דוגמא CLEAN UP (בשילוב בדיקה לנקות והכנה הכלאה) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש YM-30 Microcon עמודה: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוספת 100 Cot-1 μL DNA (1μg/μL) לעמודה. אל תיגע קרום עם טפטפת בקצה. </li><li style=";text-align:right;direction:rtl"> בריכת התגובות (התייחסות לדוגמה) ולהוסיף עמודה. </li><li style=";text-align:right;direction:rtl"> המקום עמודה צינור סיפק ספין ב g 13,000 במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> הוספת 200 H מזוקקים μL 2 0 עד קרום וחזור ספין לשטוף. </li><li style=";text-align:right;direction:rtl"> בטל צינור ולהוסיף 45 פתרון הכלאה μL: (רוש DIG קל) </li></ul> אופציונלי: להוסיף זרע 5μL טעון DNA הרינג (יחידת 10μg/μL, Promega) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> היפוך Microcon, מקום צינור שכותרתו חדשים, ספין על 3000 גרם במשך 3 דקות. </li></ul></li></ol> חישוב INCORPORATIONS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר 1.5 μL ו Fluorophore למדוד ההתאגדות באמצעות ספקטרופוטומטר NanoDrop. באמצעות המאגר הכלאה שלך ריק (DIG קל) בצע את ההוראות על המסך. (ניתן לשחזר את הדוגמה הכן, במידת הצורך, לאחר מדידה). (הערה:. Incorporations מתחת 3.0 pmol / μL בערוץ גם הראו התוצאות משתנה) </li><li style=";text-align:right;direction:rtl"> לפגל על ​​85 מעלות צלזיוס למשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> מקום בדיקה ב 45 ° C במשך שעה 30 דקות עד 1 (מאפשר Cot-1-DNA חישול). </li></ol> ARRAY הכלאה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 44 מקום פתרון μL בדיקה על coverslip (22 מ&quot;מ x 60 מ&quot;מ) (פישר סיינטיפיק). אם זמין, המקום coverslip בשקופית חם או לחסום חום טרום חימם עד 45 ° C כדי לשמור על הטמפרטורה. </li><li style=";text-align:right;direction:rtl"> יישר להחליק על coverslip פתרון בדיקה, נמוך קצה אחד של שקופיות המאפשר מגע עם הפתרון הכלאה. המשך נמוך עד coverslip מצורף לשקופית עם מתח הפנים. היפוך שקופיות. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופית לתוך קלטת הכלאה (Telechem), טרום חימם עד 45 ° C, ולהוסיף 10 μL מים החריץ התחתון (כדי לשלוט לחות במהלך הכלאה). </li><li style=";text-align:right;direction:rtl"> חותם קלטת ו דגירה של 36-40 שעות ב 45 ° C. </li></ol> ARRAY כביסה הערה: כל פתרונות לשטוף הם ב-pH 7.0 הסרה של השקופית מתוך הקלטת ההכלאה היא קריטית. DIG קל במהירות מתגבש בטמפרטורת החדר. שקופית צריך להיות שקוע מיד את הפתק מכסה הסיר בפתרון לשטוף. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוספת כ 60 מ&quot;ל של 0.1XSSC, 0.1% SDS (pH 7.0, 45 ° C) לשטוף פתרון בצנצנת Coplin לפני פתיחת תא הכלאה. </li><li style=";text-align:right;direction:rtl"> תא פתוח, להוסיף שקופית לשטוף ולהסיר פתרון coverslip (הוא אמור להחליק). </li><li style=";text-align:right;direction:rtl"> לשטוף את השקופית 3 פעמים 0.1XSSC + SDS 0.1% ב 45 ° C למשך דקה 1 כל אחד עם תסיסה. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את השקופית 3 פעמים 0.1XSSC טריים *. לא צריך להיות שום בועות שיורית מן לשטוף את SDS גלוי. * שקופיות יכול להישאר הפתרון הסופי עד לשטוף מוכן צנטריפוגות ולסרוק (~ 15 דקות). </li><li style=";text-align:right;direction:rtl"> צנטריפוגה את השקופית 50 צינורות מ&quot;ל פלקון 700 גרם ב 3 דקות. </li><li style=";text-align:right;direction:rtl"> מיד לסרוק את השקופיות (עוצמות אות תפחת לאורך זמן.) </li></ol>

<ol>
	<li>Lockwood, W. W., Chari, R., Chi, B., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+array+comparative+genomic+hybridization+technologies+and+their+applications+in+human+genetics.">Recent advances in array comparative genomic hybridization technologies and their applications in human genetics.</a> European Journal of Human Genetics. 14, 139-1x`48 (2006).</li><li>Ishkanian, A. S., Malloff, C. A., Watson, S. K., deLeeuw, R. J., Chi, B., Coe, B. P., Snijders, A., Albertson, D. G., Pinkel, D., Marra, M. A., Ling, V., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiling+resolution+DNA+microarray+with+complete+coverage+of+the+human+genome.">A tiling resolution DNA microarray with complete coverage of the human genome.</a> Nature Genetics. 36, 299-303 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Ishkanian, A. S., Malloff, C. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+high+throughput+validation+of+amplified+fragment+pools+of+BAC+DNA+for+constructing+high+resolution+CGH+arrays.">Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.</a> BMC Genomics. 5, 6 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Horsman, D. E., Squire, J. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetically+balanced+translocations+are+associated+with+focal+copy+number+alterations.">Cytogenetically balanced translocations are associated with focal copy number alterations.</a> Human Genetics. 120, 795-805 (2007).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeGH+--+a+software+tool+for+visualization+of+whole+genome+array+comparative+genomic+hybridization+data.">SeeGH -- a software tool for visualization of whole genome array comparative genomic hybridization data.</a> BMC Bioinformatics. 5, 13 (2004).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., Ng, R. T., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MD-SeeGH:+a+platform+for+integrative+analysis+of+multi-dimensional+genomic+data.">MD-SeeGH: a platform for integrative analysis of multi-dimensional genomic data.</a> BMC Bioinformatics. 9, 243 (2008).</li><li>Coe, B. P., Ylstra, B., Carvalho, B., Meijer, G. A., Macaulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+resolution+of+array+CGH.">Resolving the resolution of array CGH.</a> Genomics. 89, 647-653 (2007).</li><li>Garnis, C., Coe, B. P., Lam, S. L., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+array+CGH+increases+heterogeneity+tolerance+in+the+analysis+of+clinical+samples.">High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.</a> Genomics. 85, 790-793 (2005).</li></ol>

DNA באיכות ירודה לא תספק פרופיל הכלאה טובה. זה חיוני כדי להבטיח כי המדגם ו-DNA הפניה ללא מזהמים כגון פנול, RNA, מלח, וכו &#39;שעשויים להפריע צעד תיוג אקראי הממשלה לפני תחילת ניסוי הכלאה. לדוגמה resuspension של דנ&quot;א טריס - EDTA (TE) במקום מים אינה מומלצת כמו ריכוז מלח גבוה יכולים לעכב את התגובה תיו...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5X Klenow Buffer</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Random Octamers </td>
<td>Reagent</td>
<td>Alpha DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X dNTP mix</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>2mM dATP, dGTP, dTTP, 1.2mM dCTP</td>
</tr>
<tr>
<td>Cy-3 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Genomic DNA Reference</td>
<td>Reagent</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>Example of a possible reference</td>
</tr>
<tr>
<td>Klenow</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>YM-30 Column</td>
<td>Reagent</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cot-1 DNA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG Easy</td>
<td>Reagent</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sheared Herring Sperm DNA</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>22mm x 60mm</td>
</tr>
<tr>
<td>Hybridization Cassette</td>
<td>Tool</td>
<td>Telechem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

technical demonstration of whole genome array comparative genomic hybridization

גנומית השוואתי מערך ההכלאה (מערך CGH) היא שיטה לגילוי הרווחים וההפסדים של מגזרים דנ&quot;א או מינון גנים הגנום 1. ההתקדמות בטכנולוגיה זו אפשרו השוואה ברזולוציה גבוהה של הגנום כולו לזיהוי שינויים גנטיים בסרטן ומחלות גנטיות אחרות 2. תת Megabase ריצוף שנקבע בהחלטה מערך (או SMRT) מערך מורכב של קבוצה של כ 30,000 חופפים כרומוזום בקטריאלי מלאכותי (BAC) שיבוטים כי ההיקף הגנום האנושי בזוג ~ kilobase 100 (KB) 2 קטעים. מטרות אלה BAC מסונתזים בנפרד הבחין בשני עותקים על זכוכית שקופית אחת 2-4. Array CGH מבוססת על העיקרון של הכלאה תחרותי. לדוגמה ו-DNA התייחסות מסומנים באופן דיפרנציאלי עם Cyanine-3 ואת צובעת Cyanine-5 ניאון, ושיתוף הכלאה למערך. לאחר תקופת דגירה הדגימות מאוגד נשטפים משקופית ואת מערך היא הדמיה. חבילת זמין בחינם תוכנות מותאמות אישית בשם SeeGH (www.flintbox.ca) משמש כדי תהליך נפח גדול של נתונים שנאספו - ניסוי אחד מייצר 53,892 נקודות נתונים. SeeGH מדמיין את האות log2 עוצמת היחס בין 2 דגימות בכל מטרה BAC אשר מיושרים אנכית עם עמדת כרומוזומליות 5,6. מערך SMRT יכול לזהות שינויים קטנים כמו 50 KB בגודל 7. מערך SMRT יכול לזהות מגוון של סידור אירועים DNA לרבות רווחים DNA, הפסדים ומחיקות amplifications הומוזיגוטים. יתרון ייחודי של המערך SMRT היא שאף אחד יכול להשתמש DNA שבודד פורמלין דגימות פרפין קבוע מוטבע. בשילוב עם דרישות קלט נמוך של ה-DNA unamplified (25 100ng) זה מאפשר אפיון של דגימות יקרות כמו אלה המיוצרים על ידי microdissection 7,8. זו מיוחסת גודלו של כל יעד הכלאה BAC המאפשר קשירה של דגימות שכותרתו מספיק כדי לייצר אותות לצורך זיהוי. יתרון נוסף של הפלטפורמה הזו היא סובלנות ההטרוגניות של רקמות, הקטנת הצורך microdissection רקמות משעמם 8. זה פרוטוקול וידאו הדרכה צעד אחר צעד מהתווית DNA קלט דרך לאותת הרכישה עבור מערך שלם ריצוף הגנום נתיב SMRT.

Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

סרט הווידאו הזה הוא הפגנה טכני של פרוטוקול הכלאה עבור כל ריצוף הגנום נתיב מערך CGH, שסורקת את הגנום האנושי כולו רק באמצעות 25-100 ng של DNA כי ניתן לבודד מתוך מגוון של מקורות, כולל חומר ארכיוני קבוע פורמלין.

הפגנה טכנית של הכלאה שלמות מערך הגנום הגנום השוואתי

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

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Biology

12.7K Views.  BC Cancer Research Centre. *The slides can remain in the 

Video: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

הר שלם כפול באתרו הכלאה של עוברים צ&#39;יק מוקדם

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

דג הזברה שלמים הר ברזולוציה גבוהה פעמיים פלורסנט באתרו הכלאה

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

שימוש הר שלם באתרו הכלאה קישור לביולוגיה מולקולרית האורגניזם

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarrays DNA: בקרת איכות המדגם, הכלאת מערך סריקה ו

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

הר שלם<em&gt; באתר</em&gt; הכלאה של E8.5 לעוברי E11.5 עכבר

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

מסכי הגנום תחרותי של ספריות שמרים המבורקדים

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

תפוקה גבוהה באתרו הכלאה שיטה לאפיון דפוסי ביטוי mRNA של העכבר עוברית בדרכי השתן ואברי המין התחתון

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

זיהוי גנטי של וריאנטים נדירים מן הרצף נקווה שימוש Splinter

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

הר כולו RNA פלורסנט<em&gt; באתר</em&gt; הכלאה של<em&gt; דרוזופילה</em&gt; עוברים

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.