We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. בדיקה הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג oligonucleotides מרובה הייחודי בXist (oligonucleotides 20-30 נוקלאוטיד באורך, טמפרטורת 63-65 מעלות צלזיוס היתוך, טבלת 1) עם שינוי 5&#39;-אמינו ולהשעות במים. </li><li style=";text-align:right;direction:rtl"> כמויות בריכת equimolar של oligonucleotides עם 5&#39;-אמינו שינוי (סה&quot;כ 4.5 מיקרוגרם) ותווית עם צבע פלואורסצנטי אמין-reactive הבא הוראות היצרן. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים את oligonucleotides נקווה ב5 μl הסופי של מים nuclease חינם ולהוסיף 3 μl של סודיום ביקרבונט 1 M לפתרון oligonucleotide. </li><li style=";text-align:right;direction:rtl"> הוספת DMSO 2 μl לבקבוקון של צבע אמין-reactive ובקצרה מערבולת. </li><li style=";text-align:right;direction:rtl"> מערבבים את פתרון oligonucleotide עם צבע תגובתי בDMSO דגירה את התערובת בטמפרטורת חדר למשך שעה 1 בחושך. </li></ol></li><li style=";text-align:right;direction:rtl"> לטהר את בדיקות oligonucleotide שכותרתו fluorescently על ידי עמודת G-25 ואחריו משקעים אתנול. <ol style=";text-align:right;direction:rtl"><li&gt; טור צנטריפוגה G-25 ב 750 XG עבור 1 דקות כדי להסיר חיץ אחסון עודף. </li><li style=";text-align:right;direction:rtl"> מוסיף מים nuclease ללא 40 μl לתערובת התיוג מיוצרת בשלב 1.2.3 ואחול על עמודת G-25. צנטריפוגה ב 750 XG למשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף 50 מים μl nuclease ללא, 10 נתרן אצטט μl 3 M (pH 5.2) ו -250 אתנול μl לבדיקה המטוהרת מהשלב 1.3.2. חנות ב -80 מעלות צלזיוס למשך 30 דקות ו צנטריפוגות ב 21,000 XG במשך 15 דקות על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> שטוף את oligonucleotides זירז פעם אחת עם 1 מיליליטר של אתנול 70%. </li><li style=";text-align:right;direction:rtl"> להשעות מחדש ב -50 μl nuclease ללא מים או TE (10 מ&quot;מ טריס-HCl, pH 7.5, 1 mM EDTA) לריכוז סופי של כ -10 מיקרומטר. </li></ol></li><li style=";text-align:right;direction:rtl"> לדלל את בדיקות oligonucleotide שכותרתו fluorescently לריכוז סופי של nM 250 עם חיץ הכלאה (פוראמיד 10%; 2x SSC [300 מ&quot;מ NaCl; ציטראט 30 מ&quot;מ נתרן]; 2 מ&quot;ג / מיליליטר BSA, 10% סולפט dextran). הערה: עם פרוטוקול FISH זה מהר, p oligonucleotideגלימות בXist תוכננו והשתמשו בריכוז גבוה מפרוטוקול FISH הרגיל באמצעות בדיקות oligonucleotide כדי לקצר את זמן הדגירה של הכלאה. </li></ol> 2. החלק הכנה הערה: ניתן להכין שקופיות לאימונו-FISH באמצעות עכבר גזע עוברי (ES) או גוף embryoid (EB) הבחנה תאים 20, 21 או על ידי גידולם ישירות בשקופית שטופל פולי-ליזין או באמצעות cytospin עם השעיה תא. כאן, הכנת שקופיות על ידי cytospin מוצגת. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב את מדיום התרבות בצלחת 6 באר. לשטוף עם PBS (בטמפרטורת חדר), ולשאוב ביסודיות. </li><li style=";text-align:right;direction:rtl"> הוסף 0.5 מיליליטר של טריפסין לצלחת 6-גם, דגירה למשך 5-10 דקות ב 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הוסף 5 מיליליטר של מדיום ולפזר תאים על ידי pipetting בעדינות מעלה ומטה מספר פעמים. </li><li style=";text-align:right;direction:rtl"> העבר את התאים לצינור 15 מיליליטר, צנטריפוגות במשך 5 דקות ב XG 200 בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> מחק את המדיוםד בעדינות להשעות את התאים 2 מיליליטר PBS. </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות hemocytometer, ולדלל השעיה תא ב 4 x 10 5 תאים / מיליליטר ידי PBS. </li><li style=";text-align:right;direction:rtl"> להרכיב שקופיות, כרטיס מסנן וקאמרי עם קליפ ולהכניס אותם לתוך החריצים המתאימים ברוטור של cytospin. הוסף 250 μl של כל דגימה מוכנה בשלב 2.6 לכל תא של היחידות שהתכנסו בcytospin. צנטריפוגה ב 1500 סל&quot;ד במשך 10 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> מלאו 3 צנצנות coplin על קרח עם 40 מיליליטר כל אחד מחיץ PBS, CSK קר כקרח (10 צינורות מ&quot;מ, pH 6.8; 100 מ&quot;מ NaCl; 300 סוכרוז מ&quot;מ; 3 מ&quot;מ MgCl 2), וחיץ CSKT (חיץ CSK עם 0.5% Triton X-100), בהתאמה. </li><li style=";text-align:right;direction:rtl"> לאחר cytospin הוא סיים, להעביר במהירות את השקופית לתוך PBS קר כקרח ודגירה של 5 דקות. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית למאגר CSK קר כקרח ודגירת דקות 1. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית למאגר CSKT קר כקרח ודגירה של 5 דקות. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית בחזרה לbuf CSK קר כקרחFER דגירה דקות 1. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית לתוך paraformaldehyde 4% ב- ​​PBS ודגירה של 10 דקות בטמפרטורת חדר. </li></ol> 3. Immunofluorescence <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את השקופיות בצנצנת coplin עם PBST (1x PBS עם Tween-20 0.1%) לכמה שניות. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופיות בשיפוע המאפשר נותר חיץ לזרום במורד המגלשה. לנגב בזהירות נוזל עודף מהשקופיות ולמקם אותו בתיבת pipet-קצה ריק עם מים. כיסוי השקופית עם 40 μl של פתרון חסימה (1x PBS, BSA 1%, 20-Tween 0.1%), מניח בעדינות coverslip, ודגירה של 10 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> הסר את coverslip, להסיר את פתרון החסימה, ולהוסיף 40 μl של הנוגדן הראשוני היסטון H3K27me3 המדולל לריכוז של 1: 500 בחסימת פתרון. מניחים את coverslip חזרה על שקופיות דגירה במשך 30 דקות בטמפרטורת חדר. הערה: מעכב RNase בפתרון החסימה עשויה להיות מועילה כדי לשמר RNA בRNA FIS זהפרוטוקול H כאשר נוגדני polyclonal משמשים. </li><li style=";text-align:right;direction:rtl"> לשטוף להחליק 2 פעמים בצנצנות coplin מלאות PBST, במשך 5 דקות כל אחד, בעדינות רועדת בזמן הכביסה. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופיות בשיפוע המאפשר נותר חיץ לזרום במורד המגלשה. לנגב בזהירות נוזל עודף ובמקום להחליק בחזרה לתוך תיבת הקצה. שכבה עם 40 μl של הנוגדנים משני (1: 500) מדוללים בחסימת פתרון, במקום coverslip, ודגירה במשך 15 דקות בחושך. לשלבים הבאים, לשמור על השקופיות בחושך בכל העת. </li><li style=";text-align:right;direction:rtl"> לשטוף 2 פעמים בCoplin צנצנות עם PBST כמו בשלב 3.4. </li></ol> FISH RNA 4. עם oligonucleotide בדיקות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טרום חם תיבת pipet-קצה ריק עם מים בתחתית כדי למנוע שקופיות מהתייבשות על 37 מעלות צלזיוס במשך FISH RNA בשלב 4.3. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופיות בשיפוע המאפשר נותר חיץ לזרום במורד המגלשה. לנגב בזהירות את נוזל עודף השקופיות מצעד 3.6 ובמקום להחליק לתוך תיבת pipet-הקצה עם ואטאה בתחתית. הוסף 500 μl של חיץ לשטוף FISH (פוראמיד 10% ב2x SSC) בשקופית ודגירה של 5 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> הסר פתרון לשטוף FISH, להוסיף 8 μl של בדיקות oligonucleotide לשקופית, במקום להחליק כיסוי, ולהעביר את השקופיות לתיבת pipet-טיפ מראש חימם עם מים בתחתית; לדגור על 37 מעלות צלזיוס במשך 1-2 שעות. </li><li style=";text-align:right;direction:rtl"> מניחים 3 צנצנות coplin מראש חימם באמבט מים ב 37 ° C עם חיץ לשטוף FISH, חיץ לשטוף FISH עם DAPI, ו2x SSC. </li><li style=";text-align:right;direction:rtl"> לאחר הדגירה שעה 1-2 לRNA FISH, לשים את השקופיות לחיץ לשטוף FISH מראש חימם. </li><li style=";text-align:right;direction:rtl"> דגירה השקופיות במשך כמה דקות עד coverslip נופל של השקופית. </li><li style=";text-align:right;direction:rtl"> מניחים את השקף חזרה לחיץ לשטוף, ולהמשיך דגירה במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית לצנצנת coplin אחרת עם חיץ לשטוף FISH מראש חימם עם 0.5 ng / ml DAPI ודגירה של 5 דקות. </li><li style=";text-align:right;direction:rtl"> העבר את השקופית לצנצנת coplin אחרת עם מראש חימם 2x SSC ודגירה של 5 ק&quot;מn. </li><li style=";text-align:right;direction:rtl"> מניחים את השקופיות בשיפוע המאפשר נותר חיץ לזרום במורד המגלשה. לנגב בזהירות נוזל עודף ובמקום להחליק בחזרה לתוך תיבת קצה יבשה. הוסף 4 μl של antifade עם DAPI לשקופית ומניח coverslip. </li><li style=";text-align:right;direction:rtl"> לאטום את coverslip עם לק ולהתבונן תחת מיקרוסקופ. השקופית יכולה להיות מאוחסן על 4 מעלות צלזיוס בחושך במשך כמה חודשים. </li></ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+RNAs+mark+sex+chromosomes+in+stem+cells.">Telomeric RNAs mark sex chromosomes in stem cells.</a> Genetics. 182, 685-698 (2009).</li><li>Raj, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+individual+endogenous+RNA+transcripts+in+situ+using+multiple+singly+labeled+probes.">Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.</a> Methods Enzymol. 472, 365-386 (2010).</li><li>Kimura, H., Hayashi-Takanaka, Y., Goto, Y., Takizawa, N., Nozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+histone+H3+modifications+as+revealed+by+a+panel+of+specific+monoclonal+antibodies.">The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies.</a> Cell Struct Funct. 33, 61-73 (2008).</li><li>Ogawa, Y., Sun, B. K., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersection+of+the+RNA+interference+and+X-inactivation+pathways.">Intersection of the RNA interference and X-inactivation pathways.</a> Science. 320, 1336-1341 (2008).</li><li>Kohlmaier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromosomal+memory+triggered+by+Xist+regulates+histone+methylation+in+X+inactivation.">A chromosomal memory triggered by Xist regulates histone methylation in X inactivation.</a> PLoS Biol. 2, 171 (2004).</li><li>Wang, K. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+long+noncoding+RNAs.">Molecular mechanisms of long noncoding RNAs.</a> Mol Cell. 43, 904-914 (2011).</li></ol>

No conflicts of interest declared.

במאמר זה, שהצגנו פרוטוקול חיסוני FISH מהיר שלוקח פחות מ 5 שעות כדי להשלים הכנת שקופיות, Xist RNA FISH, וimmunofluorescence לH3K27me3. בהשוואה לגישה חיסונית FISH הכללי לXist RNA זיהוי, אשר בדרך כלל צריך דגירה הלילה לRNA FISH, פרוטוקול זה לא רק מפחית באופן משמעותי את משך זמן, אלא גם משפר את הרגישות של ח?...

איון כרומוזום X יונקים (xci) הוא אסטרטגיה כדי לפצות על חוסר האיזון במינון גן צמוד X בין XX וXY, שבו אחד משני כרומוזומי X-בנקבות הוא תעתיק 1 מומת. איון כרומוזום X הוא מערכת מודל נהדרת עבור RNA קידוד שאינו ארוך מחקר (lncRNA). איון כרומוזום X מוסדר על ידי lncRNAs מרובה, ונחקר בהרחבה בעשרות השנים האחרונות לחשוף את מנגנוני crosstalk בין lncRNAs, שעתוק, מבנה הכרומטין וארגון גרעיני 2, 3. מרכז איון X (XIC) הממוקם על כרומוזום X הוא מוקד גנטי מורכב מורכב ממספר גני ייצור RNAs ללא קידוד 4. X-פעיל תמליל ספציפי (Xist) lncRNA ביונקי eutherian הוא lncRNA אחד כזה אשר ממלא תפקיד מכריע באיון כרומוזום X 5,6. תמלילי Xist מקיפים את מיקומו של פוture Xi ליזום איון כרומוזום X, ומופיע כענן כאשר דמיינו באמצעות RNA FISH; מערך זה נקרא &quot;Xist הענן&quot; 7. מאז Xist RNA אינטראקציה עם אנזימי שינוי הכרומטין שונים, שיתוף לוקליזציה של ענני Xist עם שינויים אפיגנטיים שונים להכרומטין השקט ושעתוק דיכוי הוא ציין במהלך איון כרומוזום X 8. לדוגמא, Xist RNA אינטראקציה עם מורכב Polycomb מדכא 2 (PRC2) האחראית על H3K27me3 וגורם למצב הכרומטין דיכוי 9. המופע של ענן Xist על Xi ושיתוף הלוקליזציה שלה עם שינוי H3K27me3 האינטנסיבי מייצג נוף heterochromatin פקולטטיביים של Xi 10,11. טכניקות ציטוגנטית, כגון DNA / RNA FISH יש כברת דרך מהשיטה המסורתית באמצעות בדיקות radiolabelled 12 לטכניקות האחרונות ומתקדמות ברגישות ובמשופרותהדמיה ניאון באמצעות בדיקות oligonucleotide מרובות 13,14. FISH DNA / RNA בשילוב עם immunofluorescence נעשה שימוש באופן שגרתי ככלי ציטולוגית להבין ארגון spatiotemporal גרעיני, לוקליזציה RNA, מבנה הכרומטין ושינויים. הכנת הבדיקה סטנדרטית ביותר עבור RNA FISH כרוך בשימוש בפלסמיד או כרומוזום מלאכותי שיבוטים חיידקים (BAC) והתיוג הבא שלהם או עם תרגום לכינוי או לתחולו אקראי 15. עם זאת, כמעט 70% מהגנים בעכברים ו -40% מהגנים בבני האדם להראות חפיפה של תמלילי תחושה וantisense 16, ומכאן צורך בשיטת FISH גדיל הספציפי כדי להבדיל תמלילי תחושה וantisense. במבחנה בדיקות RNA עיבד (riboprobe ) משמש לעתים קרובות לFISH גדיל RNA ספציפי 17,18; עם זאת, מדובר בהכנת שיבוט פלסמיד או מוצר PCR עם T7, SP6, או אמרגן T3 וriboprobes סינתזה. יתר על כן, Riboprobes נגזר מgenomic DNA או cDNA לעתים קרובות מכיל אזורים שאינם ספציפיים ואלמנטים חוזרים, אשר יביאו לרעש רקע גבוה. בעיה נוספת היא שriboprobes, שהם כמה מאה נוקלאוטידים באורך ומכיל fluorophores מרובה, לא יכולה לחדור ביעילות לתוך הגרעין. כדי לעקוף את זה, בדיקות oligonucleotide קצרות מרובות שכותרתו עם fluorophore אחד בסוף פותחו שיש לי רגישות טובה, עוצמת אות אחידה, וקלות לניקוי וטיפול 14. בנוסף, oligonucleotides DNA הוא בדרך כלל יציב יותר מRNA. אנחנו מוחלים אסטרטגיה דומה של שימוש בoligonucleotides Xist RNA FISH 19 עם immunofluorescence להבין את הדינמיקה אפיגנטיים של Xi הנגרמת על ידי Xist lncRNA במהלך התהליך של איון כרומוזום X. פרוטוקול זה מתאר את הקמתה של בדיקות oligonucleotide והכנה נכונה של תאים, כמו גם ניצול של immunofluorescence וRNA FISH. Xist RNA FISH באמצעות oligonucle מרובהotides היא גישה יעילה עלות בטווח הארוך אם Xist RNA FISH מבוצע באופן שוטף במעבדתו של אחד. טכניקה זו יכולה לשמש כדי לזהות lncRNAs בתאים תוך מיפוי שיתוף הלוקליזציה שלה עם שינויים או גורמים אפיגנטיים בו-זמנית. אחד יתרונות עיקריים של הפרוטוקול הוא היכולת לשנות אותו בקלות כדי להתאים תחומי המחקר של אחד.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">oligonucleotides with 5&#8217;-amine modification</td>
			<td style="width:187px;">IDT</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Alexa Fluor 488 Reactive Dye</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A32750</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MicroSpin G-25 columns</td>
			<td style="width:187px;">GE Healthcare</td>
			<td style="width:119px;">27-5325-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cytospin 2</td>
			<td style="width:187px;">Shandon</td>
			<td align="right" style="width:119px;">59900102</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">10715859103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Histone H3K27me3 antibody</td>
			<td style="width:187px;">Active Motif</td>
			<td style="width:119px;">61017</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Accutase</td>
			<td style="width:187px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">A21424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ProLong Gold antifade reagent</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">P36931</td>
		</tr>
	</tbody>
</table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

שילוב ניאון RNA הכלאה באתר (דגים) עם immunofluorescence (חיסוני דגים) יוצר בטכניקה שיכולה להיות מועסק ברמת התא הבודד כדי לזהות את הדינמיקה המרחבית של לוקליזציה RNA עם תובנה סימולטני ללוקליזציה של חלבונים, שינויים אפיגנטיים ופרטים נוספים אשר יכול להיות מודגש על ידי immunofluorescence. איון כרומוזום X הוא פרדיגמה להשתקת גנים ארוכה RNA קידוד שאינו (lncRNA) בתיווך. הצטברות X-פעיל תמליל ספציפי (Xist) lncRNA (נקראת Xist ענן) באחת משני כרומוזומים X-בנקבות יונקים היא שלב קריטי ליזום איון כרומוזום X. RNA Xist במישרין או בעקיפין אינטראקציה עם אנזימי שינוי הכרומטין שונים ומציג נופים אפיגנטיים ברורים לכרומוזום X פעיל (Xi). סימן היכר ידוע אפיגנטיים אחד Xi הוא trimethyl ליזין היסטון H3 27 שינוי (H3K27me3). כאן אנו מתארים חיסוני FISH פשוט ומהירפרוטוקול לאיתור Xist RNA באמצעות RNA FISH עם בדיקות oligonucleotide מרובות בשילוב עם immunofluorescence של H3K27me3 לבחון את הלוקליזציה של Xist RNA ושינויים אפיגנטיים הקשורים. שימוש בתוצאות בדיקות oligonucleotide בזמן דגירה קצר יותר וגילוי רגיש יותר של Xist RNA בהשוואה לבדיקות RNA במבחנה עיבד (riboprobes). פרוטוקול זה מספק כלי רב עוצמה להבנת הדינמיקה של lncRNAs והשינוי אפיגנטיים הקשורים אליו, מבנה הכרומטין, ארגון גרעיני ורגולצית תעתיק.

נציג תמונות של חיסוני FISH מהיר מוצגות באיור 1 א. שיתוף לוקליזציה של אות הענן וH3K27me3 Xist RNA על Xi התגלתה בהבחנת תאים נקביים. ביום 12 על בידול, יותר מ -90% מתאי EB היו ענן Xist (איור 1). בדיקות oligonucleotide הקצר חדרו יעילות לתוך הגרעינים, שמובילות להדמיה של כמעט כל אותות H...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

המהיר פלורסנט<em&gt; באתר</em&gt; הכלאת פרוטוקול לXist RNA משולב עם Immunofluorescence של היסטון שינוי באיון כרומוזום X

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

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Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.8K Views.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Video: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

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 ...

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Non-רדיואקטיבי באתרו הכלאה פרוטוקול ישים עבור נורווגיה Spruce ומגוון של מיני צמחים

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

פלואורסצנציה באתרו הכלאה (FISH) פרוטוקול של זרע אדם

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

ב הכלאה באתרו עבור לוקליזציה המדויק של תמלילי בצמחים

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

זיהוי של רנ&quot;א נגיפי על ידי הקרינה<em&gt; באתרו</em&gt; הכלאה (דגים)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

ניתוח של שעתוק גנים מהתא הבודד על ידי RNA פלורסנט<em&gt; באתר</em&gt; הכלאה (דגים)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

עיתוי מעתיק כרומוזום בשילוב עם פלורסנט<em&gt; באתר</em&gt; הכלאה

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

רנ&quot;א<em&gt; באתר</em&gt; הכלאה בעוברי הר שלמים והיסטולוגיה הסלולרית מותאמת לElasmobranchs הימי

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...