ברצוננו להודות לאיימי רגן לקריאה מדוקדקת של כתב היד. עבודה זו נתמכה על ידי מענק NGFN פלוס # 01GS0801, MRC מלגת מענק G1002523 וNHSBT מענק WP11-05 לLD וDFG מענק FR2938/1-1 לCCF

<ol>
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Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources.</a> Biotechniques. 19, 942-945 (1995).</li><li>Friedel, C. C., D&#246;lken, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+tagging+and+purification+of+nascent+RNA:+implications+for+transcriptomics.">Metabolic tagging and purification of nascent RNA: implications for transcriptomics.</a> Mol. Biosyst. 5, 1271-1278 (2009).</li><li>Friedel, C. C., Kaufmann, S., D&#246;lken, L., Zimmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HALO+-+A+Java+framework+for+precise+transcript+half-life+determination.">HALO - A Java framework for precise transcript half-life determination.</a> Bioinformatics. , (2010).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

תיוג חילוף חומרים של RNA החדש שעבד באופן משמעותי מגביר את העצמה של טכנולוגיות תפוקה גבוהה כמו microarrays וRNA-Seq על ידי מתן תבניות מתאימות יותר להתייחס לשאלה הביולוגית של עניין. הפרוטוקול הנוכחי עבר אופטימיזציה נרחבת. היא מאפשרת העשרה&gt; 1,000 פי חדש של RNA עיבד ומספקת תוצאות לשח...

פרופיל גנטי ביטוי הוא כלי מרכזי המשמש ללמוד תהליכים תאיים וברשת יחסי הגומלין המורכבת המשויכת. שפע מחקרים על mRNA היו בדרך כלל בשיטה של ​​בחירה כדי להשיג תובנות בסיסיות לתוך המנגנונים המולקולריים שבבסיס. הפיתוח של microarrays השלם transcriptome 1, ולאחרונה רצף של הדור הבא, של רנ&quot;א (RNA-אילך) 2-4 מונע את הגישה הזו. בעוד טכנולוגיות אלה חוללו מהפכה ההבנה של המורכבות של ביטוי גני הסלולר שלנו שהם עומדים בפני מגבלות עיקריות בשל מאפיינים פנימיים של תבנית המדגם שלהם, כלומר סך הסלולרי RNA. שינויים ראשונים, לטווח קצר ברמות רנ&quot;א הכל אינם תואמים שינויים בשערי שעתוק, אבל מטבעם תלויים ברנ&quot;א מחצית החיים של התמלילים, בהתאמה. בעוד אינדוקציה פי חמישה מתמליל, קידוד לדוגמה קצר לגורם שעתוק, תהיה בקלות להבחין ברנ&quot;א הכלבתוך שעה, אותו הגיוס של תמליל, למשל קידוד חיים ארוכים לאנזים המטבולי, יישאר כמעט בלתי נראה. בנוסף, גם שלם כיבוי (&gt; למטה רגולציה של פי 1,000) בשיעור השעתוק של גן ממוצע עם RNA מחצית חיים של חמש שעות תהיה פשוט לקחת חמש שעות לרמות רנ&quot;א הכל לירידה של רק כפולה . לכן, ניתוח של RNA הכולל מעדיף את זיהוי של עד הוויסות של תמלילים קצרים ימים, שרבים מהם לקודד לגורמי שעתוק וגנים עם פונקציות רגולטוריות 5. בנוסף, המפל קינטית הנכון של רגולציה הוא מוסתר ולא יכול להיות מובחן אירועי איתות ראשוניים משני. שניהם, בתורו, עלולים לגרום להטיה משמעותית בניתוחים ביואינפורמטיקה במורד הזרם. שנית, לא ניתן לייחס שינויים ברמות RNA הכולל לשינויים בסינתזה או ריקבון רנ&quot;א. מדידות של זה האחרון דורשות גישות פולשניות סלולריים, כגון חסימת transcriptiעל שימוש actinomycin 6 ד &#39;, וניטור מורחב של ריקבון מתמשך RNA לאורך זמן. עם ה-mRNA ממוצע מחצית חיים בתאי יונקים של 5 - 10 שעות 5,7, רמות ה-mRNA של רוב הגנים תהיה ירדו רק על ידי פחות מכפול לאחר כמה שעות של מעצר תעתיק. הבדלים קטנים ולא אלו לגרום למדידות לא מדויקות בעליל של mRNA מחצית חיים עבור רוב הגנים סלולריים בשל אופי מעריכי של משוואות המתמטיות המשמשות כבסיס. לבסוף, בעוד RNA-ואילך בסך הכל הסלולרי RNA גילה כי כמחצית מהגנים שלנו כפופה לאירועי שחבור חלופיים 8, קינטיקה הבסיסית, כמו גם את המנגנונים הדינמיים מנחים רגולציה רקמות והקשר ספציפי של RNA עיבוד יישאר הבין היטב. בנוסף, התרומה של רנ&quot;א עיבוד לביטוי ההפרש גן, במיוחד עבור RNAs ללא קידוד, עדיין לא נקבע. בסך הכל, על המגבלות האלה מייצגות מכשולים עיקריים לדוגמנות הקינטית bioinformatic של המנגנונים המולקולריים שבבסיס. לאחרונה פתחנו גישה, פרופיל גנטי ביטוי ברזולוציה גבוה המכונה, כדי להתגבר על בעיות אלה 5,7,9. היא מבוססת על תיוג חילוף חומרים של RNA החדש שעבד באמצעות 4-thiouridine (4sU תיוג), נגזרת uridine מתרחש באופן טבעי, ומספקת גישה ישירה לתמלילים חדשים שעבדו עם התערבות מינימאלית בצמיחת תאים וביטוי גנים (ראה איור 1) 5, 10-12. חשיפה של תאים אוקריוטים לתוצאות 4sU בספיגתו המהירה, זירחון 4sU-אדנוזין, והכללתו RNA החדש שעבד. בעקבות בידוד בסך הכל הסלולרי רנ&quot;א, רנ&quot;א שבריר 4sU שכותרתו הוא biotinylated תיאול-במיוחד ליצירת קשר דיסולפיד בין ביוטין ואת הרנ&quot;א חדש שעבד. לאחר מכן ניתן להפריד &#39;סה&quot;כ סלולרי RNA&#39; כמותית לכותרתו (&quot;זה עתה עיבד &#39;) וללא תווית (&#39; מראש existinרנ&quot;א) &quot;G עם טוהר גבוה באמצעות חרוזים מגנטיים streptavidin מצופים. לבסוף, שכותרתו RNA הוא התאושש מן החרוזים פשוט על ידי הוספת סוכן הפחתה (למשל dithiothreitol) ביקוע אג&quot;ח דיסולפיד ומשחרר את הרנ&quot;א חדש שעבד מהחרוזים. RNA עיבד חדש מתאר את פעילות תעתיק של כל גן במהלך פרק הזמן של חשיפת 4sU. 4sU תיוג בלוח הזמנים של דקות ובכך מספק תמונת תמונת מצב של ביטוי אוקריוטים גן ותבנית אידיאלית לניתוחי bioinformatic מורד זרם (לדוגמה: ניתוח אמרגן). במקרים בהם ניתן להניח תנאי מצב יציב, את היחס של זה עתה עיבד / סך הכל, שזה עתה עיבד / ללא תווית וללא תווית / RNA הכולל לספק גישה לא פולשנית לרנ&quot;א המדויק מחצית חיים 7,13. בנוסף, חשוב לציין כי RNA החדש שעבד מטוהר לאחר קטן כמו 5 דקות של 4sU תיוג (5 דקות 4sU-RNA) הוא צעיר מ -15 ו -60 דקות 4sU-RNA.בעת ביצוע שני אולטרה קצר וארוך יותר בהדרגה 4sU תיוג בהגדרה ניסיונית אחת בשילוב עם RNA-ואילך, קינטיקה של רנ&quot;א עיבוד מתגלה ברזולוציה של נוקלאוטיד 9. לבסוף, ניתוחים בזמן קורס של RNA חדש שעבד ומוחלט בשילוב עם מודלים חישוביים לאפשר ניתוח אינטגרטיבי של סינתזה וריקבון 14-RNA. לסיכום, גישה זו מאפשרת ניתוח הישיר של הדינמיקה של סינתזת RNA, עיבוד, והשפלה בתאים אוקריוטים. זה ישים בכל אורגניזמים המודל הגדולים, כולל יונקים, חרקים (דרוזופילה), דו חיים (Xenopus), ושמרים 5,15,16. זה תואם באופן ישיר עם microarray ניתוח 5,17, RNA-9,13,14 ואילך, והוא ישים in vivo 12,15. הנה, אנחנו פירוט המתודולוגיה לתייג, לבודד, ולטהר את RNA החדש שעבד בתאי יונקים בתרבית. בנוסף, potentiבעיות ואת החסרונות al הם דנו.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>4-thiouridine</td>
 <td>Carbosynth</td>
 <td>T4509</td>
 <td>Prepare 50 mM stock in sterile H2O, store at -20 &deg;C in aliquots of 50-500 &mu;l, discard unused reagent, do not refreeze.</td>
 </tr>
 <tr>
 <td>Trizol</td>
 <td>Invitrogen</td>
 <td>15596026 (100 ml), 15596018 (200 ml)</td>
 <td>WARNING - CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma</td>
 <td>372978</td>
 <td>WARNING - HAZARDOUS TO HEALTH</td>
 </tr>
 <tr>
 <td>Isopropanol</td>
 <td>Sigma</td>
 <td>650447</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate, nuclease-free</td>
 <td>Sigma</td>
 <td>C8532</td>
 <td>Prepare 1.6 M stock solution using nuclease-free water.</td>
 </tr>
 <tr>
 <td>5M nuclease-free NaCl</td>
 <td>Sigma</td>
 <td>71386</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>Nuclease-free H2O</td>
 <td>Sigma</td>
 <td>W4502</td>
 <td>Make 1 ml aliquots in nuclease-free tubes.</td>
 </tr>
 <tr>
 <td>RNA precipitation buffer</td>
 <td></td>
 <td></td>
 <td>1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under strictly nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma</td>
 <td>459844</td>
 <td>Use with nuclease-free water to prepare 80% ethanol, store at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>1 M nuclease-free Tris Cl, pH 7.5</td>
 <td>Lonza</td>
 <td>51237</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>500 mM nuclease-free EDTA, pH 8.0</td>
 <td>Invitrogen</td>
 <td>15575-020</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>10x Biotinylation Buffer (BB)</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water, make aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Dimethylformamide (DMF)</td>
 <td>Sigma</td>
 <td>D4551</td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-Link biotin-HPDP</td>
 <td>Pierce</td>
 <td>21341</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Phase Lock Gel Heavy tubes 2.0 ml</td>
 <td>Eppendorf</td>
 <td>0032 005.152</td>
 <td>Optional for the chloroform extraction step.</td>
 </tr>
 <tr>
 <td>Zeta membrane</td>
 <td>BIORAD</td>
 <td>162-0153</td>
 <td></td>
 </tr>
 <tr>
 <td>10x Dot blot binding buffer</td>
 <td></td>
 <td></td>
 <td>100 mM NaOH, 10 mM EDTA</td>
 </tr>
 <tr>
 <td>Biotin-oligo</td>
 <td></td>
 <td></td>
 <td>5'-biotin, 25 nucleotides, any sequence</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulphate</td>
 <td>Fisher</td>
 <td>BPE9738</td>
 <td>For 100 ml 20% stock solution, add 20 g SDS to 80 ml PBS pH 7-8 and adjust volume to 100 ml. Keep all high-percentage SDS solutions above 20 &deg;C. Warm the solutions slightly should SDS precipitate.</td>
 </tr>
 <tr>
 <td>EZ-Link Iodoacetyl-LC-Biotin</td>
 <td>Pierce</td>
 <td>21333</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg iodoacetyl-biotin in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml. Generates irreversible, thiol-specific biotinylation.</td>
 </tr>
 <tr>
 <td>Phosphate buffer saline</td>
 <td>Gibco</td>
 <td>10010-015</td>
 <td></td>
 </tr>
 <tr>
 <td>Dot blot blocking buffer</td>
 <td></td>
 <td></td>
 <td>Mix 20 ml 20% SDS with 20 ml 1 x PBS pH 7-8 and add EDTA to the final concentration of 1 mM.</td>
 </tr>
 <tr>
 <td>Streptavidin-horseradish peroxidase </td>
 <td>Vector Laboratories</td>
 <td>SA5004</td>
 <td>Store at -20 &deg;C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 &mu;l Streptavidin-HRP before use. </td>
 </tr>
 <tr>
 <td>ECL reagent</td>
 <td>GE Healthcare</td>
 <td>RNP2109</td>
 <td>Use following the manufacturer's instructions.</td>
 </tr>
 <tr>
 <td>Super RX, X-RA Film, 18x24 cm</td>
 <td>Fujifilm</td>
 <td>47410 19236</td>
 <td></td>
 </tr>
 <tr>
 <td>&mu;Macs Streptavidin Kit</td>
 <td>Miltenyi</td>
 <td>130-074-101</td>
 <td>Store the beads at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P1379</td>
 <td></td>
 </tr>
 <tr>
 <td>Washing buffer</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O.</td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>Sigma</td>
 <td>43817</td>
 <td>Prepare as 100 mM DTT in nuclease-free H2O, always prepare fresh before use.</td>
 </tr>
 <tr>
 <td>RNeasy MinElute Kit</td>
 <td>Qiagen</td>
 <td>74204</td>
 <td>Store columns at 4 &deg;C, remaining components of the kit at room temperature.</td>
 </tr>
 <tr>
 <td>1.5 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.692.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>2.0 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.694.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>50 ml tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>All solutions/reagents should be stored at room temperature unless otherwise specified.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>UV/VIS spectrophotometer</td>
 <td>Thermo Scientific</td>
 <td>NanoDrop 1000</td>
 <td>Or equivalent. Use low volume (1-2 &mu;l) for measurements of low RNA concentrations to avoid excessive sample loss.</td>
 </tr>
 <tr>
 <td>Polypropylene 15 ml centrifuge tubes</td>
 <td>VWR International</td>
 <td>525-0153</td>
 <td>In contrast to standard 15 ml tubes, these tolerate up to 15,000 &times; g</td>
 </tr>
 <tr>
 <td>High-speed centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-25</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>High-speed rotor</td>
 <td>Beckman Coulter</td>
 <td>JLA-16250</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Adaptors for 15 ml tubes</td>
 <td>Laborger&auml;te Beranek</td>
 <td>356964</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Refrigerated table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5430 R</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Thermomixer</td>
 <td>Eppendorf</td>
 <td>Thermomixer compact</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Magnetic stand</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-109</td>
 <td>One stand holds 8 &mu;Macs columns.</td>
 </tr>
 <tr>
 <td>Waterbath</td>
 <td>Grant</td>
 <td>SUB Aqua 5</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Ultra-fine scale</td>
 <td>A&amp;D</td>
 <td>GR-202</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel iBase Power System</td>
 <td>Invitrogen</td>
 <td>G6400UK</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel EX 1% agarose precast gels</td>
 <td>Invitrogen</td>
 <td>G4020-01</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 </tbody>
</table>

metabolic labeling of newly transcribed rna for high resolution gene expression profiling of rna synthesis, processing and decay in cell culture

הפיתוח של microarrays השלם transcriptome והדור הבא של רצף יש מהפכה ההבנה של המורכבות של ביטוי גני הסלולר שלנו. יחד עם הבנה טובה יותר של המנגנונים המולקולריים המעורבים, מידות מדויקות של קינטיקה הבסיסית הפכו להיות חשובה יותר ויותר. כאן מתודולוגיות רבי עוצמה אלה, עומדות בפני מגבלות עיקריות בשל מאפיינים פנימיים של דגימות תבניתו הם לומדים, RNA הסלולרי כלל כלומר. במקרים רבים שינויים ברנ&quot;א הסלולרי כלל להתרחש גם לאט מדי או מהר מדי כדי לייצג את האירועים המולקולריים שבבסיס וקינטיקה שלהם עם רזולוציה מספיקה. בנוסף, התרומה של שינויים בסינתזת רנ&quot;א, עיבוד, וריקבון אינה מובחנות בקלות. לאחרונה פיתחנו פרופיל גנטי ביטוי ברזולוציה גבוה כדי להתגבר על המגבלות האלה. הגישה שלנו מבוססת על תיוג חילוף חומרים של RNA החדש שעבד עם 4-thiouriלסעוד (ובכך גם המכונה 4sU תיוג) ואחריו טיהור קפדני של RNA עבד מחדש באמצעות biotinylation תיאול ספציפי וחרוזים מגנטיים streptavidin מצופים. זה ישים למגוון רחב של יצורים בעלי חוליות, לרבות תסיסנית, ושמרים. אנחנו מיושמים בהצלחה 4sU תיוג ללמוד קינטיקה בזמן אמת של פעילות גורם שעתוק, לספק מדידות מדויקות של RNA מחצית חיים, ולקבל תובנות לתוך רומן קינטיקה של רנ&quot;א עיבוד. לבסוף, יכולים להיות מועסקים מודלים חישוביים ליצירת ניתוח משולב, מקיף של המנגנונים המולקולריים שבבסיס.

1. חומר מוצא ותשואות צפויות בעקבות 1 שעה (HR) של 4sU-RNA החדש שעבד חשיפה מייצג כ 4 - 1% מכלל RNA הסלולרי. זה יהיה נמוך יותר ב- נעצר גדילת תאים כפי שהם כבר לא לסנתז RNA לחשבון לצמיחת תאים / שכפול. כאשר תיוג עבור שעה 1, אנו ממליצים להתחיל את assay עם 60-80 מ...

Watch this Scientific Journal Video about Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture at JoVE.com

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

סה&quot;כ הסלולרי RNA מספק תבנית עניים לחקר שינויים קצרי טווח בסינתזה וריקבון RNA, כמו גם את הקינטיקה של רנ&quot;א עיבוד. כאן, אנו מתארים תיוג חילוף חומרים של RNA החדש שעבד עם 4-thiouridine אחרי biotinylation תיאול ספציפי ולטיהור של RNA החדש שעבד ומאפשר להתגבר על המגבלות האלה.

סימון מטבולי של רנ&quot;א חדש עיבד לביטוי פרופיל גנטי רזולוציה גבוהה של הסינתזה רנ&quot;א, עיבוד וריקבון בתרבית תאים

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

metabolic-labeling-newly-transcribed-rna-for-high-resolution-gene

Research

JoVE Journal

Biology

27.0K Views.  Max von Pettenkofer Institute. סה"כ הסלולרי RNA מספק תבנית עניים לחקר שינויים קצרי טווח בסינתזה וריקבון RNA, כמו גם את הקינטיקה של רנ"א עיבוד. כאן, אנו מתארים תיוג חילוף חומרים של RNA החדש שעבד עם 4-thiouridine אחרי biotinylation תיאול ספציפי ולטיהור של RNA החדש שעבד ומאפשר להתגבר על המגבלות האלה.

Video: Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

RNA סינתזה ניתוק מן דג הזברה cDNA עובריים עבור ניתוח ביטוי גנים

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

אינדוקציה ובדיקה של היפוקסיה בתרבות התא

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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 ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

מטבולית תיוג של אוצין עשיר חזור קינאזות 1 ו -2 עם רדיואקטיבי פוספט

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

מדידה של קצב חילוף החומרים ב<em&gt; דרוזופילה</em&gt; באמצעות Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

ממוקד RNA רצף Assay לאפיון ביטוי גנים ושינויים הגנום

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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 ...