SV הוא אסירת תודה על התמיכה במימון K22AI077353 NIH וקרן Landenberger. KR מודה בתודה מימון מ-NIH (5SC1GM083754).

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יש המחברים אין לחשוף.

התגובה במבחנה &#39;מראש mRNA 3 מחשוף, שבוצעה בתמציות גרעיניות תא הלה או עם גורמי מחשוף מופרדים מתמציות אלה, אפשרה זיהוי של גורמי מחשוף הליבה והמכלולים העיקריים שלהם 16-21. לאחרונה חלבונים רבים יותר הקשורות לגורמים אלה זוהו 22, והתגובה במבחנה יכולה להמשיך ולשפוך אור על כמה חלבונים אלה תורמים לתגובה. אולי בגלל התגובה in vivo נראית 23 cotranscriptional, או משום שכמה גורמים תורמים עלולים ללכת לאיבוד בעת חילוץ ודיאליזה, היעילות היא לעתים קרובות עני, ורק לעתים נדירות היא מחשוף יותר מ 30% מושגת. בנוסף, יעילות התגובה יכולה פתאום לרדת מיום ליום ללא כל סיבה נראית לעין. לכן, חשוב להעריך שצעדים הם הקריטיים ביותר, במיוחד כאשר מנסים להתאים את התגובה לcel הלה שונה או באופן ויראלי הנגועls, לסוגי תאים אחרים או למצעים חדשים. ללא ספק, את האיכות של התמצית ואת הרעננות של במבחנה RNA עיבד הן בעל חשיבות עליונה, כפי שהוא צריך לעבוד תחת בקפדנות תנאי RNase חינם. בריאותם של התאים בזמן שהם נקצרים להפקה היא גם חשובה. התאים אמורים להופיע בריא, הם צריכים להיות הכפלה בפחות מ 24 שעות, הם צריכים להיות באו מתקרבים שלב יומן באמצע, ולא צריך להיות כמה תאים מתים או פסולת בלתי מוסברת אחרת. ברור, לא צריכים להיות מזוהמים התאים על ידי Mycoplasma או חיידקים אחרים. המצע לא צריך להיעשות עם פעילות ספציפית גבוהה מדי, כמו זה יכול להוביל להידרדרות נראית לעין. כאשר מספיק רצף גדול משני צדיו של אתר המחשוף נחשב, וpolyadenylation החלופי מטופלת, מספר פולי השונים (א &#39;) אותות רצף בגנום האנושי ללא ספק עולה על מספרם של גנים 24 </sעד&gt;. כמו שיש השתנות ב&quot; הכוח &quot;של אותות שונים שנבדקו, כאשר התאמת ההליך לסוג תא חדש, עדיף להתחיל עם אחד מאותות polyadenylation החזק, כגון וירוס הקופי לעתים קרובות נעשה שימוש 40 ( SV40) פולי ז&quot;ל () אות 25. השילוב של פולי חלשים () רצף אות עם תאים אחרים מאשר תאי הלה סטנדרטיים יכול להוכיח מתסכל, וזה יותר טוב ראשון שהשתמש במצע חזק עם תאים חדשים, או תאי הלה ללא שינוי עם מצע פולה פוטנציאל חלש. גם אחרי כל כך הרבה שנים של שימוש מוצלח, יש עדיין תעלומות קטין הקשורים לתגובת המחשוף במבחנה &#39;3. לדוגמא, מדוע הוא קריאטין פוספט צורך? הוכח כי הסיבה המקורית להכללתו - לחדש את בריכת ה-ATP - אינה חוקית 26. מעניין, זה יכול להיות מושמט עם אובדן חלקי בלבד של פעילות, כאשר תמציות גרעיניותבשימוש, אך הופך להיות יותר ויותר לפי הצורך לחלץ הוא מופרד לגורמי מחשוף מטוהרים באופן חלקי המשמשים כדי לשקם את התגובה. למעשה, phosphocholine, עוד מולקולה קטנה עם קבוצת פוספט ואמין טעונים חיובי, אבל סביר להניח שאין לו תפקיד בvivo בתגובה, כבר מצאה להיות יעיל יותר מאשר קריאטין פוספט, לפחות בתגובה מחדש 27. PVA הוא מרכיב נוסף דרישה שאינו מוסבר במלואו. הנחה הוא להיות סוכן צפיפות, מה שמוביל לעלייה אפקטיבית בריכוז גורם מחשוף, אבל סוכנים מצטופפים אחרים, כמו פוליאתילן גליקול, לא עובדים כמעט גם כן. הבנת גורמים אלה עשויות להוביל ליעילות משופרת, מה שמאפשר הרחבה של השיטה ליעילה פחות סוגי תאים ומצעי RNA, ועשויה להניב רמזים איך הגורמים השונים לעבוד.

ביוסינתזה של רוב RNAs הבוגר אוקריוטים הודעה (mRNAs) דורשת מספר שינויים לאחר תעתיק כגון מכסת, שחבור וpolyadenylation. שינויים אלה הם בדרך כלל בשילוב כדי להבטיח עיבוד נכון 1 ומאוד להגדיל את היציבות של mRNA. 3 &#39;היווצרות סוף מראש mRNAs יונקים מופקת על ידי מחשוף endonucleolytic של RNA המתהווה אחריו תוספת של שאריות adenylate ל5&#39; ביקע מוצר על ידי פולי פולימראז (א) (PAP) 2-4. ביונקים, מחשוף מושגת על ידי חלבון מורכב multicomponent, של כ 800 kDa, אשר מרכיב על רצפים מראש RNA ספציפיים. פולי () רצף אות, AAUAAA רצף hexanucleotide הקנונית השמור ביותר, מכוונים את אתר המחשוף בכ 10-30 NT במורד הזרם. אתר זה מוכר במיוחד על ידי גורם המחשוף וpolyadenylation הסגוליות (CPSF) ומקטע KD 73 שלCSPF מכיל את פעילות endonuclease. גורם גירוי המחשוף (CstF) נקשר יותר מנוון רצף אלמנט GU-U או עשיר במורד הזרם של פולי (א &#39;) באתר. נדרשו גם למחשוף הוא גורם היונקים המחשוף אני (CFIm), וגורם היונקים המחשוף השני (CFII). CFIm נקשר אתרי UGUA (N) הספציפיים באלמנטים במעלה הזרם רצף (שימושים) שהוגדרו עבור מספר גנים ונראה שהם מעורבים בתהליכים פיסיולוגיים חשובים 5-8. במבחנה, תגובות עיבוד RNA מנותחות בדרך כלל על ידי השימוש במצעי RNA רדיואקטיבי 9-12. אלה עשויים להיות מסונתזים על ידי שעתוק נגר מT7 אמרגן bacteriophage או SP6. כאשר לומדים אתר polyadenylation שלא אפיין בעבר, יש צורך להשתמש בהדנ&quot;א הגנומי ולא cDNA כדי ליצור את מצע RNA, כרצפים במורד הזרם חשובים לא יכולים להיות נוכחים בcDNA. מצעי עיצוב לכלול לפחות 150 upstrea NTמ &#39;ו50 NT במורד הזרם מאתר המחשוף / סיום על mRNA הבוגר. מוצר המחשוף נודד מהר יותר מאשר המצע; עם זאת, בגלל שברים אחרים עלולים להיווצר על ידי פעולת nuclease שאינו ספציפית, את הספציפיות של התגובה צריכה להיות מאומתת על ידי תלותה ברצפי עיבוד אותות הנכונים. לכן, מצעי RNA עם מוטציה נקודתית ברצף AAUAAA (למשל AAGAAA) ישמשו כביקורת שלילית לתגובת המחשוף. בהתחשב בעובדה שכמות קטנה של RNA רדיואקטיבי משמשת לתגובות המחשוף, RNases הווה בשפע הגבוה ביותר בתמציות הגרעיניות יכולה להיות בעייתית, ולהגביל את הבחירה של החומר המוצא להכנת תמצית. תאי הלה נוטים להכיל רמות נמוכות של RNases אנדוגני, ובכך לבצע גם במבחנים אלה. מחשוף endonucleolytic של מצעי RNA in vivo ו במבחנה ומייד אחריו פולי (א) בנוסף לכך, ה &#39;ים ביניים ביקע אינם נוכחים בכמויות לזיהוי. לכן, כדי ללמוד או רצף RNA ספציפי או חלבונים מעורבים בתגובת מחשוף, ניסויים נעשים בתנאים המונעים polyadenylation התרחשות. אין תלות של מחשוף בpolyadenylation, או להיפך, ולכן אחד לא יכול לעצור polyadenylation מבלי לפגוע בתגובת המחשוף. לפיכך, ה-ATP מוחלפת אנלוגי שרשרת סיום שחסרת ​​קבוצת הידרוקסיל &#39;3, כך שרק נוקלאוטיד יחיד יכול להיות משולב באתר פולי () ורק RNA ביקע ניתן לאתרם. בהתחשב במורכבות וברמה גבוהה של ייחוד של סוג זה של assay, אנו מתארים פרוטוקול וידאו מפורט ללמוד מחשוף endonucleolytic ידי מכונות מחשוף / midi () של מבשרי ה-mRNA במבחנה. אנו מתארים כיצד להכין תמציות גרעיניות המוסמכות, לייצר מצעי RNA רדיואקטיבי, לבצע את תגובת הביקוע, ולנתח ולפרש את התוצאהמוצרי ing. איור 1 מציג דוגמא של RNAs מצע קידוד לסוף &#39;3 ​​של HIV-1 מראש mRNAs לשמש לassay מחשוף. סוף &#39;3 ​​של הגנום RNA HIV מורכב מהרבה רצפים רגולטוריים חשובים כגון אתר פולי (א&#39;), אזור עשיר G + U, ואת אלמנט שימוש, אשר כולם הכרחיות להתבגרות יעילה של תעתיקי mRNA הנגיפי 13 . בדוגמא זו שהיינו מצפה מצע RNA הקלט להיות 338 NT ועל מחשוף 237 NT. אם polyadenylation הורשה להתרחש, כתם של מוצרים יהיה נצפה בין 237 ו437 NT.

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			<td height="21" style="height:21px;width:216px;">10x TAE</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP13354</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x TBE</td>
			<td style="width:141px;">Ameresco Life Sciences</td>
			<td style="width:119px;">0658-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP169-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Persulfate</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Acetate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A637-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40% 19:1 Acrylamide:Bis-acrylamide</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">10 901 393 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100% Absolute Ethanol 200 Proof</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acid Phenol-Chloroform</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">9720</td>
			<td>For RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintilation Fluid</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">SX18-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase Inhibitor</td>
			<td style="width:141px;">Promega</td>
			<td style="width:119px;">N261B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly (Vinyl alcohol) PVA</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Phosphate</td>
			<td style="width:141px;">Calbiochem</td>
			<td style="width:119px;">2380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100mM dATP</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP2560-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">23042-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP1700-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable Sequencing Unit</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351881-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binder Clips</td>
			<td style="width:141px;">Office Depot</td>
			<td style="width:119px;">838-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx42cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352543</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx22cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35252-7</td>
			<td>1SET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20cmx42cm Aluminum Cooling Plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352667</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx22cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35230-6</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx42cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352314-1</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35195-4</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351962</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351970</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Repel Coating</td>
			<td style="width:141px;">C.B.S. Scientific</td>
			<td style="width:119px;">SGR-0401</td>
			<td>or SGR-0101 for individual bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Loading Tips</td>
			<td style="width:141px;">Rainin</td>
			<td style="width:119px;">GT-10-4</td>
			<td>0.1-10uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sequencing PowerPac HV</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">PowerPac HV</td>
			<td>5000V/500mA/400W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Dryer Model 583</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">Model 583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrotech Vacuum Pump for gel dryer</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glogos II Glow-in-the-dark Markers</td>
			<td style="width:141px;">Agilent</td>
			<td style="width:119px;">420201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 8x10</td>
			<td style="width:141px;">Midsci</td>
			<td style="width:119px;">BX810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 14x17</td>
			<td style="width:141px;">Phenix</td>
			<td style="width:119px;">F-BX1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoradiography Cassette</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">FBCA 1417</td>
			<td>8x10 size available</td>
		</tr>
	</tbody>
</table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

סוף &#39;3 של mRNAs היונקים לא נוצר על ידי הפסקה פתאומית של שעתוק על ידי RNA פולימראז II (RNPII). במקום זאת, RNPII מסנתז mRNA המבשר מעבר לסוף RNAs הבוגר, ותהליך פעיל של פעילות endonuclease נדרש באתר ספציפי. מחשוף של RNA המבשר בדרך כלל מתרחש 10-30 NT במורד הזרם מאתר הקונצנזוס הפולה (AAUAAA) לאחר dinucleotides קליפורניה. חלבונים ממתחם המחשוף, חלבון מורכב multifactorial של כ 800 kDa, להשיג פעילות nuclease הספציפית הזה. רצפי הרנ&quot;א ספציפיים מעלה או במורדו של האתר הפולה לשלוט על הגיוס של מתחם המחשוף. מייד לאחר מחשוף, טרום mRNAs הם polyadenylated ידי פולימראז הפולה (PAP) כדי לייצר להבשיל הודעות RNA יציבים. עיבוד של סוף &#39;3 של תעתיק הרנ&quot;א ניתן ללמוד באמצעות תמציות גרעיניות סלולריות עם מצעי RNA רדיואקטיבי ספציפיים. לסיכום, 32 ארוכים &lt;/ Sup&gt; RNA מבשר uncleaved P-שכותרתו הודגרו עם תמציות גרעיניות במבחנה, ומחשוף נבחנים על ידי ג&#39;ל אלקטרופורזה וautoradiography. כאשר מחשוף ראוי מתרחש, קצר יותר 5 &#39;ביקע מוצר מזוהה וכימות. כאן, אנו מתארים assay המחשוף בפירוט משתמשים, כדוגמא, עיבוד הסוף &#39;3 של HIV-1 mRNAs.

תוצאות נציג של assay מחשוף של פולי RNA () אתרים של HIV-1 (איור 2). אנחנו יכולים לראות את מצע RNA uncleaved, המהווה את הלהקה הנודדת האיטית ביותר בחלק העליון של הג&#39;ל. המוצר ביקע הספציפי הוא להקת שבר קצרה יותר האינטנסיבית ביותר שפועלת מהר יותר בג&#39;ל בגודל הצפוי, ודווקא נעדר מassay המחשוף של mutpoly () שליטה המכיל מוטציה נקודתית ברצף הפולה RNA (mutPolyA) מצע. מוצרים פגומים של מצע הקלט עשויים לעתים להיות שנצפו. כימות של פעילות המחשוף עשויה להיקבע על ידי עלילת densitometric של היחס uncleaved לרנ&quot;א ביקע מנורמל של RNA uncleaved 100%. <img alt="איור 1" fo:content-width="6in" src="/files/ftp_upload/51309/51309fig1highres.jpg" width="600" /> תיאור איור 1. סכמטי של-HIV מראשמצעים &#39;mRNA 3 סוף העיבוד, ומוצרים צפויים של במבחנה תגובות מחשוף וpolyadenylation. אתר המחשוף, dinucleotide-CA-, טמון 25 NT במורד הזרם מAAUAAA האתר הפולה. אזורי LTR: U3 (ייחודי 3 &#39;רצף), R (רצף חוזר ונשנה), וU5 (ייחודי 5&#39; ברצף). <img alt="איור 2" fo:content-width="5in" src="/files/ftp_upload/51309/51309fig2highres.jpg" width="500" /> איור 2. () פוליפוני מחשוף אתר (). 32 RNAs P-שכותרת המכיל פולי () של HIV-1 ומוטציה נקודתית של פולי () האות שמבטל מחשוף (mutPolyA) הודגרו בתמציות גרעיניות של תאי הלה-S3 או BSA כביקורת שלילית. חץ מודגש מציין 5 מוצר &#39;ביקע. הבר &#39;3 לעתים קרובות בורח ג&#39;ל או נפגע ולא תמיד נראה לעין. עלילת densitometric של פעילויות המחשוף (ב&#39;)ty מבוטא כיחס של uncleaved לרנ&quot;א ביקע מנורמל של RNA uncleaved 100%. <table border="0" cellpadding="0" cellspacing="0" width="1032" style=";text-align:right;direction:rtl"><tbody><tr><td> חיץ (100 מיליליטר) </td><td> 10 מ&quot;מ טריס (pH 7.9), 1.5 מ&quot;מ MgCl 2, 10 מ&quot;מ KCl, dithiothreitol 0.5 מ&quot;מ (DTT). </td><td> הוספת DTT רק לפני שימוש חיץ במהלך החילוץ. הוסף 1 μl של 1 M DTT לכל 1 מיליליטר של החיץ א </td></tr><tr><td> החיץ C (50 מיליליטר) </td><td> 20 מ&quot;מ טריס (pH 7.9), 25% (v / v) גליצרול, 0.42 M NaCl, 1.5 מ&quot;מ MgCl 2, חומצת 0.2 מ&quot;מ ethylenediaminetetraacetic (EDTA), 0.5 מ&quot;מ DTT, פלואוריד phenylmethylsulfonyl 0.5 מ&quot;מ (PMSF). </td><td> הוספת לוח מעכבי פרוטאז קוקטייל בוקר של עקירות. </td></tr><tr><td> החיץ D 50 (2-4 ליטר) </td><td> 20 מ&quot;מ טריס (pH7.9), 20% (v / v) גליצרול, 0.2 mM EDTA, 0.5 מ&quot;מ DTT, אמוניום סולפט 50 מ&quot;מ. </td><td> HEPES-KOH ניתן להחליף לטריס חיץ, C ו-D </td></tr><tr><td&gt; RNA הצפת elution </td><td> אצטט amonium 0.5 M, 10 מ&quot;מ MgCl 2, 1 mM EDTA, 0.2% נתרן גופרתי dodecyl (SDS) </td><td> לאחסן בטמפרטורת חדר. </td></tr><tr><td> תחנה חוצץ (ETS) </td><td> 10 מ&quot;מ טריס (pH8.0), 10 mM EDTA, 0.5% SDS </td><td> לאחסן בטמפרטורת חדר. </td></tr><tr><td> הצפת טוען מחשוף </td><td> פוראמיד 90%, 5 מ&quot;מ EDTA pH8, 0.025% כחולים bromophenol </td><td> חנות ב -20 ° C. </td></tr></tbody></table> טבלת 1. הרכב של מאגרים.

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA פולימראז II מסנתז RNA מבשר שמשתרע מעבר לקצה &#39;3 של ה-mRNA הבוגרת. סוף RNA הבוגר מופק cotranscriptionally, באתר מוכתב על ידי רצפי הרנ&quot;א, באמצעות פעילות endonuclease של מתחם המחשוף. הנה, אנחנו פירוט את השיטה ללמוד תגובות מחשוף במבחנה.

ניתוח של תגובות RNA עיבוד באמצעות מערכות חינם ניידים: 3 &#39;סוף המחשוף של Pre-mRNA מצעים<em&gt; במבחנה</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

12.7K Views.  The Scripps Research Institute.  RNA פולימראז II מסנתז RNA מבשר שמשתרע מעבר לקצה '3 של ה-mRNA הבוגרת. סוף RNA הבוגר מופק cotranscriptionally, באתר מוכתב על ידי רצפי הרנ"א, באמצעות פעילות endonuclease של מתחם המחשוף. הנה, אנחנו פירוט את השיטה ללמוד תגובות מחשוף במבחנה. 

Video: Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

וידאו ביואינפורמטיקה ניתוח גדילה אנושי בתאי גזע עובריים קולוני

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

ויזואליזציה של תיקון MG53 בתיווך קרום התא באמצעות In vivo ו במבחנה מערכות

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

יחיד קרא מותאמים mRNA-Seq סוף Illumina ספריות מ -10 ננוגרם RNA סך הכל

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

בתעתיק במבחנה תקרת של mRNA בלוציפראז Gaussia ואחריו transfection Cell הלה

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

ניתוח ספציפי של תא<em&gt; ארבידופסיס</em&gt; עלים באמצעות מיון תא הופעל פלואורסצנטי

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

דור מצע לEndonucleases של מערכות CRISPR / קאש

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

מערכות ביטוי חופשי הנייד מבוסס הלה לביטוי של<em&gt; Plasmodium</em&gt; Rhoptry חלבונים

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

תא Assay חינם לחקר הכרומטין Decondensation בסוף מיטוזה

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

זיהוי של חלבונים קושרי RNA על ידי<em&gt; במבחנה</em&gt; RNA הנפתח ב adipocyte תרבות

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.