ونود أن نشكر إيمي ريغان لقراءة متأنية للمخطوطة. وأيد هذا العمل من قبل NGFN زائد منحة # 01GS0801، MRC زمالة منحة G1002523 وNHSBT منحة WP11-05 إلى LD وDFG منحة FR2938/1-1 إلى CCF

<ol>
	<li>Brown, P. O., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+new+world+of+the+genome+with+DNA+microarrays.">Exploring the new world of the genome with DNA microarrays.</a> Nature Genetics. 21, 33-37 (1999).</li><li>Mortazavi, A., Williams, B. A., Mccue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nature Methods. 5, 621-628 (2008).</li><li>Nagalakshmi, U., Wang, Z., 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=The+transcriptional+landscape+of+the+yeast+genome+defined+by+RNA+sequencing.">The transcriptional landscape of the yeast genome defined by RNA sequencing.</a> Science. 320, 1344-1349 (2008).</li><li>Wilhelm, B. T., Marguerat, S., 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=Dynamic+repertoire+of+a+eukaryotic+transcriptome+surveyed+at+single-nucleotide+resolution.">Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution.</a> Nature. 453, 1239-U1239 (2008).</li><li>D&#246;lken, L., Ruzsics, Z., 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=High-resolution+gene+expression+profiling+for+simultaneous+kinetic+parameter+analysis+of+RNA+synthesis+and+decay.">High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay.</a> RNA. 14, 1959-1972 (2008).</li><li>Yang, E., van Nimwegen, E., 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=Decay+rates+of+human+mRNAs:+correlation+with+functional+characteristics+and+sequence+attributes.">Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes.</a> Genome Res. 13, 1863-1872 (2003).</li><li>Friedel, C. C., D&#246;lken, L., Ruzsics, Z., Koszinowski, U. H., 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=Conserved+principles+of+mammalian+transcriptional+regulation+revealed+by+RNA+half-life.">Conserved principles of mammalian transcriptional regulation revealed by RNA half-life.</a> Nucleic Acids Res. 37, e115 (2009).</li><li>Nilsen, T. W., Graveley, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+eukaryotic+proteome+by+alternative+splicing.">Expansion of the eukaryotic proteome by alternative splicing.</a> Nature. 463, 457-463 (2010).</li><li>Windhager, L., Bonfert, T., 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=Ultra+short+and+progressive+4sU-tagging+reveals+key+characteristics+of+RNA+processing+at+nucleotide+resolution.">Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.</a> Genome Res. , (2012).</li><li>Melvin, W. T., Milne, H. B., Slater, A. A., Allen, H. J., Keir, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+6-thioguanosine+and+4-thiouridine+into+RNA.+Application+to+isolation+of+newly+synthesised+RNA+by+affinity+chromatography.">Incorporation of 6-thioguanosine and 4-thiouridine into RNA. Application to isolation of newly synthesised RNA by affinity chromatography.</a> Eur. J Biochem. 92, 373-379 (1978).</li><li>Cleary, M. D., Meiering, C. D., Jan, E., Guymon, R., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthetic+labeling+of+RNA+with+uracil+phosphoribosyltransferase+allows+cell-specific+microarray+analysis+of+mRNA+synthesis+and+decay.">Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay.</a> Nature Biotechnology. 23, 232-237 (2005).</li><li>Kenzelmann, M., Maertens, S., 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=Microarray+analysis+of+newly+synthesized+RNA+in+cells+and+animals.">Microarray analysis of newly synthesized RNA in cells and animals.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 6164-6169 (2007).</li><li>Schwanh&#228;usser, B., Busse, D., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473, 337-342 (2011).</li><li>Rabani, M., Levin, J. Z., 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=Metabolic+labeling+of+RNA+uncovers+principles+of+RNA+production+and+degradation+dynamics+in+mammalian+cells.">Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells.</a> Nat. Biotechnol. , (2011).</li><li>Miller, M. R., Robinson, K. J., Cleary, M. D., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TU-tagging:+cell+type-specific+RNA+isolation+from+intact+complex+tissues.">TU-tagging: cell type-specific RNA isolation from intact complex tissues.</a> Nat. Methods. 6, 439-441 (2009).</li><li>Miller, C., Schwalb, B., 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=Dynamic+transcriptome+analysis+measures+rates+of+mRNA+synthesis+and+decay+in+yeast.">Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast.</a> Mol. Syst. Biol. 7, 458 (2011).</li><li>Weintz, G., Olsen, J. V., 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=The+phosphoproteome+of+toll-like+receptor-activated+macrophages.">The phosphoproteome of toll-like receptor-activated macrophages.</a> Mol. Syst. Biol. 6, 371 (2010).</li><li>Lipsett, M. 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+isolation+of+4-thiouridylic+acid+from+the+soluble+ribonucleic+acid+of+Escherichia+coli.">The isolation of 4-thiouridylic acid from the soluble ribonucleic acid of Escherichia coli.</a> Journal of Biological Chemistry. 240, 3975-3978 (1965).</li><li>Marcinowski, L., Liedschreiber, M., 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=Real-time+Transcriptional+Profiling+of+Cellular+and+Viral+Gene+Expression+during+Lytic+Cytomegalovirus+Infection.">Real-time Transcriptional Profiling of Cellular and Viral Gene Expression during Lytic Cytomegalovirus Infection.</a> PLoS Pathog. 8, e1002908 (2012).</li><li>Chomczynski, P., Mackey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+technical+reports.+Modification+of+the+TRI+reagent+procedure+for+isolation+of+RNA+from+polysaccharide-+and+proteoglycan-rich+sources.">Short technical reports. 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>

يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

وضع العلامات الأيضية من الحمض النووي الريبي المحولة حديثا يعزز بشكل كبير من قوة تكنولوجيات عالية الإنتاجية مثل ميكروأرس وRNA وما يليها من خلال توفير قوالب أكثر ملاءمة لمعالجة هذه المسألة البيولوجية من الفائدة. خضع هذا البروتوكول الأمثل واسعة النطاق. لأنها تتيح تخصيب...

التنميط الجيني التعبير هي أداة رئيسية تستخدم لدراسة العمليات الخلوية وشبكة التفاعل المعقد المرتبطة بها. وكانت الدراسات على وفرة مرنا عادة الأسلوب المفضل للحصول على رؤى الأساسية في الآليات الجزيئية الكامنة. غذى تطوير ميكروأرس كامل Transcriptome على 1، وفي الآونة الأخيرة، تسلسل الجيل القادم من الحمض النووي الريبي (RNA وما يليها) 2-4 هذا النهج. في حين أن هذه التقنيات قد أحدثت ثورة فهمنا من تعقيد الخلوية التعبير الجيني، فإنهم يواجهون قيودا كبيرة بسبب الخصائص الذاتية من عينة قالب بهم، أي مجموع الخلوية الحمض النووي الريبي. التغييرات الأولى، على المدى القصير في مجموع مستويات الحمض النووي الريبي لا تتطابق التغيرات في أسعار النسخ، ولكنها تعتمد بطبيعتها على الحمض النووي الريبي نصف العمر من النصوص منها. في حين أن الاستقراء خمسة أضعاف من لم يدم طويلا نص، على سبيل المثال ترميز لعامل النسخ، وسوف يكون كشفها بسهولة في الحمض النووي الريبي مجموعفي غضون ساعة، نفس تحريض على الأجل الطويل نص، على سبيل المثال الترميز لانزيم الأيضية، وسوف تظل غير مرئية تقريبا. وبالإضافة إلى ذلك، حتى استكمال اغلاق القناة (&gt; 1،000 أضعاف أسفل تنظيم) في معدل النسخ من الجين بمتوسط ​​مع نصف عمر خمس ساعات RNA سوف ببساطة تأخذ خمس ساعات لمجموع مستويات الحمض النووي الريبي لينخفض ​​بنسبة شقين فقط . ولذلك، فإن تحليل الحمض النووي الريبي مجموع تفضل الكشف عن ما يصل التنظيم من النصوص لم يدم طويلا، وكثير منها ترميز لعوامل النسخ والجينات مع المهام التنظيمية 5. وبالإضافة إلى ذلك، يتم حجب تتالي الحركية الحقيقية للتنظيم، ولا يمكن أن تكون متباينة الأحداث يشير الأساسي من الثانوي. كلا، بدوره، قد يؤدي إلى تحيز كبير في التحليلات المعلوماتية الحيوية المصب. ثانيا، لا يمكن أن تعزى التغيرات في إجمالي مستويات الحمض النووي الريبي للتغيرات في تركيب الحمض النووي الريبي أو تسوس. قياسات لهذه الأخيرة تتطلب نهجا الغازية الخلية، على سبيل المثال حظر transcriptiحول استخدام أكتينوميسين D 6، ورصد طويلة من الاضمحلال RNA الجارية مع مرور الوقت. بمتوسط ​​مرنا نصف العمر في خلايا الثدييات من 5-10 ساعة 5،7، لن يؤدي إلا إلى فقد انخفضت مستويات مرنا من معظم الجينات بنسبة أقل من شقين بعد عدة ساعات من اعتقال النسخي. هذه الاختلافات الصغيرة بدلا يؤدي إلى قياسات غير دقيقة بشكل فاضح من مرنا نصف العمر بالنسبة لغالبية الجينات الخلوية نظرا لطبيعة الأسي من المعادلات الرياضية الكامنة. وأخيرا، بينما RNA وما يليها من الحمض النووي الريبي مجموع الخلوية كشفت أن ما يقرب من نصف جيناتنا تخضع لأحداث بديلة الربط 8، حركية الكامنة فضلا عن آليات ديناميكية توجيه الأنسجة ومحددة السياق تنظيم معالجة الحمض النووي الريبي لا تزال غير مفهومة تماما. وبالإضافة إلى ذلك، فإن مساهمة RNA تجهيز لتباين التعبير الجيني، ولا سيما بالنسبة للالرناوات غير الترميز، لا يزال يتعين تحديدها. وإجمالا، هذه القيود تمثل العقبات الرئيسية للالنمذجة المعلوماتية الحيوية الحركية من الآليات الجزيئية الكامنة. وضعنا مؤخرا نهجا، ووصف الجينات وارتفاع القرار التنميط التعبير، للتغلب على هذه المشاكل 5،7،9. لأنه يقوم على وضع العلامات الأيضية من الحمض النووي الريبي المحولة حديثا باستخدام 4-thiouridine (4sU الجغرافية)، وهو مشتق يوريدين التي تحدث بشكل طبيعي، ويوفر الوصول المباشر إلى النصوص المحولة حديثا مع الحد الأدنى من التدخل في نمو الخلايا والتعبير الجيني (انظر الشكل 1) 5، 10-12. التعرض للخلايا حقيقية النواة إلى نتائج 4sU في امتصاص على وجه السرعة، الفسفرة إلى 4sU-ثلاثي الفوسفات، وإدماجها في الحمض النووي الريبي المحولة حديثا. بعد عزل الحمض النووي الريبي مجموع الخلوية، الكسر RNA 4sU المسمى هو البيروكسيديز ثيول على وجه التحديد توليد السندات ثاني كبريتيد بين البيوتين وRNA المحولة حديثا. &#39;مجموع RNA الخلوية&#39; ثم يمكن فصل كميا في المسمى (&#39;المحولة حديثا&#39;) وتوضيحها (&quot;ما قبل existinز &#39;) RNA مع نقاء عالية باستخدام الخرز المغناطيسي streptavidin المغلفة. وأخيرا، يتم استرداد المسمى الحمض النووي الريبي من الخرز ببساطة عن طريق إضافة عامل مختزل (على سبيل المثال dithiothreitol) الشق السندات ثاني كبريتيد والافراج عن RNA المحولة حديثا من الخرز. كتب حديثا RNA يصور النشاط النسخي من كل جين خلال الإطار الزمني للتعرض 4sU. 4sU الجغرافية في مقياس الوقت من دقيقة وبالتالي يقدم صورة لقطة من حقيقية النواة التعبير الجيني وقالب مثالي لأسفل مجرى التحليلات المعلوماتية الحيوية (مثل تحليل المروج). في الحالات التي يمكن افتراض ظروف الحالة المستقرة، نسب المحولة حديثا / المجموع، كتب حديثا / الخالي من الملصقات وغير المسماة / الحمض النووي الريبي مجموع توفير وصول غير الغازية لRNA دقيق نصف حياة 7،13. وبالإضافة إلى ذلك، فمن المهم أن نلاحظ أن الحمض النووي الريبي المحولة حديثا تنقيته بعد اقل من 5 دقائق من 4sU الجغرافية (5 دقائق 4sU-RNA) هو أصغر من 15 و 60 دقيقة 4sU-RNA.عند تنفيذ كلا فائقة القصيرة والطويلة تدريجيا 4sU الجغرافية في إعداد تجريبية واحدة جنبا إلى جنب مع الحمض النووي الريبي وما يليها، وكشف حركية معالجة الحمض النووي الريبي في قرار النوكليوتيدات 9. وأخيرا، تحليلات الوقت طبعا من الحمض النووي الريبي المحولة حديثا ومجموع جنبا إلى جنب مع النمذجة الحاسوبية تسمح لتحليل تكاملية تخليق الحمض النووي الريبي والاضمحلال 14. وفي الختام، فإن هذا النهج يسمح للتحليل المباشر لديناميات توليف الحمض النووي الريبي، وتجهيز، وتدهور في الخلايا حقيقية النواة. هذا ينطبق في جميع الكائنات الحية بما فيها الثدييات نموذج رئيسي والحشرات (ذبابة الفاكهة)، البرمائيات (الضفدع)، والخميرة 5،15،16. وهو متوافق مباشرة مع تحليل ميكروأري 5،17، RNA-يليها 9،13،14، وينطبق في الجسم الحي 12،15. هنا، ونحن بالتفصيل منهجية لتسمية وعزل، وتنقية RNA المحولة حديثا في خلايا الثدييات مثقف. وبالإضافة إلى ذلك، potentiوتناقش المشاكل القاعدة والمزالق.

<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

تطوير ميكروأرس كامل Transcriptome على وتسلسل الجيل القادم قد أحدثت ثورة فهمنا من تعقيد الخلوية التعبير الجيني. جنبا إلى جنب مع فهم أفضل للآليات الجزيئية المسؤولة، أصبحت قياسات دقيقة لحركية الكامنة ذات أهمية متزايدة. هنا، تواجه هذه المنهجيات قوية قيود كبيرة بسبب الخصائص الذاتية من العينات قالب التي يدرسونها، أي الحمض النووي الريبي مجموع الخلوية. في كثير من الحالات تغيرات في الحمض النووي الريبي مجموع الخلوية تحدث إما ببطء شديد أو بسرعة كبيرة جدا لتمثيل الأحداث الجزيئية الكامنة وحركية مع القرار كافية. وبالإضافة إلى ذلك، لم يتم التمييز بينها بسهولة مساهمة تغييرات في تركيب الحمض النووي الريبي، وتجهيز، والاضمحلال. وضعنا مؤخرا عالية الدقة الجينات التنميط التعبير للتغلب على هذه القيود. ويستند نهجنا على وضع العلامات الأيضية من الحمض النووي الريبي المحولة حديثا مع 4 thiouriتناول الطعام (وبالتالي يشار إلى 4sU الجغرافية أيضا)، يليه تنقية صارمة من الحمض النووي الريبي كتب حديثا باستخدام biotinylation ثيول محددة والخرز المغناطيسي streptavidin المغلفة. وهو ينطبق على طائفة واسعة من الكائنات الحية بما فيها الحيوانات الفقارية، ذبابة الفاكهة، والخميرة. طبقنا بنجاح 4sU الجغرافية لدراسة حركية في الوقت الحقيقي من أنشطة عامل النسخ، وتوفير قياسات دقيقة من الحمض النووي الريبي نصف حياة، والحصول على رؤى جديدة في حركية معالجة RNA. وأخيرا، والنمذجة الحسابية يمكن استخدامها لتوليد تحليل متكامل وشامل من الآليات الجزيئية الكامنة.

1. انطلاق مادة وعائدات المتوقعة بعد 1 ساعة (HR) من 4sU التعرض RNA المحولة حديثا يمثل حوالي 1-4٪ من مجموع RNA الخلوية. هذا وسوف يكون أقل في الخلايا اعتقال النمو لأنها لم تعد توليف الحمض النووي الريبي لحساب لنمو الخلايا / النسخ المتماثل. عندما ?...

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

يوفر مجموع RNA الخلوية قالب الفقيرة لدراسة التغيرات قصيرة الأجل في تخليق الحمض النووي الريبي والاضمحلال وكذلك حركية معالجة RNA. هنا، نحن تصف وضع العلامات الأيضية من الحمض النووي الريبي المحولة حديثا مع 4 thiouridine تليها biotinylation ثيول محددة وتنقية RNA المحولة حديثا السماح للتغلب على هذه القيود.

وصفها الأيضية من الحمض النووي الريبي كتب حديثا للدقة عالية التنميط التعبير الجيني من تخليق الحمض النووي الريبي، ومعالجة تسوس في خلية ثقافة

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. يوفر مجموع RNA الخلوية قالب الفقيرة لدراسة التغيرات قصيرة الأجل في تخليق الحمض النووي الريبي والاضمحلال وكذلك حركية معالجة RNA. هنا، نحن تصف وضع العلامات الأيضية من الحمض النووي الريبي المحولة حديثا مع 4 thiouridine تليها biotinylation ثيول محددة وتنقية RNA المحولة حديثا السما...

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.

التحضير لزراعة الخلايا المكونة للدم الابتدائية من نخاع عظام الفئران لElectroporation

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.

عزل الحمض النووي الريبي من الجنينية التجميعي واسماك الزرد [كدنا] لتحليل الجينات

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.

باستخدام خلية مكافحة الآلي لتبسيط دراسات التعبير الجيني : ضربة قاضية سيرنا من IL - 4 التعبير الجيني في الخلايا التابعة 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.

التعريفي واختبار نقص الأكسجة في الثقافة خلية

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.

في المختبر، والسد النسخ من مرنا luciferase المراسل Gaussia تلاه ترنسفكأيشن الخلية هيلا

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.

الأيضية وصفها من لوسين ريتش كرر تحركات 1 و 2 مع المشعة الفوسفات

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.

تحليل الحمض النووي الريبي العالمي التجميعي على مستوى خلية واحدة نقص الأكسجة التالي

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.

قياس معدل الأيض في<em&gt; ذبابة الفاكهة</em&gt; باستخدام قياس التنفس

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.

استهدفت RNA تسلسل الفحص لتحديد خصائص التعبير الجيني والجينوم التعديلات

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.

الكشف عن البروتينات ملزمة الحمض النووي الريبي<em&gt; في المختبر</em&gt; RNA المنسدلة في خلية شحمية الثقافة

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 ...