Name: novel rna-binding proteins isolation by the rapid methodology
Uploaded: 2016-09-30T14:00:00
Description: Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

نشكر البروفيسور جيف Gerst وبوريس Slobodin للحصول على المشورة المفيدة في إعداد بروتوكول السريع وتوفير البلازميدات اللازمة. كما نشكر الدكتور أفيغيل عتير-انديه لمساعدتها في إنشاء هذا البروتوكول والدكتور تمار زيف من مركز البروتيوميات Smoler لمساعدتها مع تحليل LC-MS / MS. نشكر الأستاذ TG Kinzy (روتجرز) للأجسام المضادة YEF3. وأيد هذا العمل من قبل منحة 2011013 من مؤسسة الثنائية للعلوم.

ملاحظة: إدراج تسلسل يتكون من 12 موقعا ملزم MS2 (MS2 حلقات، MS2L) في موضع الجيني المطلوب، عادة بين الإطار القراءة المفتوحة (ORF) و &quot;UTR 3. ويرد بروتوكول مفصلة لهذا التكامل في أماكن أخرى 22. تحقق الإدراج الصحيح والتعبير PCR، وتحليل الشمالي أو RT-PCR 20،23. فمن المهم للتحقق من...

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أساليب مختلفة تستخدم في عزل من mRNAs محددة لتحديد البروتينات المرتبطة بها 11،34 35. وتنطبق هذه الأساليب في التجارب المختبرية واستراتيجيات الجسم الحي للتحقيق تفاعلات الحمض النووي الريبي البروتين. في المختبر طرق احتضان نسخه خارجيا RNA مع المح...

البروتينات ملزمة الحمض النووي الريبي (الممارسات التجارية التقييدية) تمثل حوالي 10٪ من س. بروتينات خميرة 1،2 وحوالي 15٪ من البروتينات الثدييات 3-5. كانوا متورطين في العديد من العمليات الخلوية مثل معالجة مرنا بعد النسخي والتنظيم، الترجمة، نشوء حيوي الريبوسوم، الحمض الريبي النووي النقال aminoacylation وتعديل، وإعادة عرض لونين، وأكثر من ذلك. فريق فرعي هامة من الممارسات التجارية التقييدية هي بروتينات ملزمة مرنا (mRNPs) 6،7. في سياق نضوج مرنا، الممارسات التجارية التقييدية مختلفة تربط نص وتوسط معالجة النووي والتصدير من النواة، توطين الخلوية والترجمة وتدهور 6-8. وهكذا، فإن مجموعة متميزة من الممارسات التجارية التقييدية منضم إلى نص معين في أي نقطة زمنية يحدد التجهيز لها، وفي نهاية المطاف مصيرها. تحديد الممارسات التجارية التقييدية المرتبطة مرنا يمكن أن تحسن بشكل كبير من فهمنا للعمليات التي يقوم تنظيمها بعد النسخي. تنوعا الوراثية، وقد استخدمت أساليب المجهرية، والكيمياء الحيوية والمعلوماتية الحيوية لتحديد البروتينات المشاركة في التنظيم مرنا (إعادة النظر في 11/9). ومع ذلك، سوى عدد قليل من هذه الأساليب تمكن من التعرف على البروتينات المرتبطة مرنا هدف معين. من المذكرة هو الخميرة ثلاثة نظام الهجين (Y3H)، الذي يستخدم مرنا من الفائدة كطعم لفحص مكتبة التعبير في خلايا الخميرة. وعادة ما تكون لاحظت الحيوانات المستنسخة إيجابية من خلال مجموعة مختارة النمو أو مراسل التعبير 12-14. والميزة الرئيسية لهذا الأسلوب هو وجود عدد كبير من البروتينات التي يمكن مسحها في بيئة الخلوية والقدرة على قياس قوة التفاعل الحمض النووي الريبي البروتين. وتشمل النواحي العدد الكبير نسبيا من نتائج إيجابية كاذبة بسبب غير محددة وملزمة، وقدرة عالية على النتائج السلبية الكاذبة الواجبة، في جزء منه، إلى misfolding فريسة البروتين الانصهار أو الحمض النووي الريبي الطعم. بديل للنهج الوراثي purifi تقاربالموجبة من الحمض النووي الريبي مع البروتينات المرتبطة بها. بولي والتي تحتوي على من mRNAs يمكن عزلها من خلال استخدام جزئية الأعمدة دينارا، والكشف عن البروتينات المرتبطة بها عن طريق قياس الطيف الكتلي. يتم حفظها تفاعل الحمض النووي الريبي البروتين في سياق الخلوي من قبل يشابك، الأمر الذي يجعل الرابطة التساهمية قصيرة المدى. استخدام العمود DT بنسبة ضئيلة غلة نظرة عالمية للبروتيوم بأكمله الذي يرتبط مع أي بولي والتي تحتوي على مرنا 3،5،15. ومع ذلك، هذا لا يوفر قائمة من البروتينات التي ترتبط مع مرنا بشكل خاص. جدا متاحة لإنجاز مثل هذا تحديد عدد قليل من الطرق. يستتبع طريقة زوج ترنسفكأيشن من الحمض النووي مع التكامل إلى الهدف مرنا 16،17. ويرد الحمض النووي أيضا إلى الببتيد، والذي يسمح يشابك إلى الممارسات التجارية التقييدية في المناطق المجاورة القريبة من موقع التفاعل. بعد يشابك، وحامض الممارسات التجارية التقييدية-الببتيد النووي يمكن عزل ويتعرضون لتحليل البروتينات. في الآونة الأخيرة، وكانت منهجية القائم على أبتمرطبقت بنجاح لمقتطفات من خطوط الخلايا الثديية 18. تم وضع أبتمر RNA مع تحسين التقارب إلى streptavidin وتنصهر لسلسلة من الفائدة (عنصر والاتحاد الافريقي الغني (ARE) في هذه الحالة). كان يعلق على أبتمر-ARE RNA لحبات streptavidin ومختلطة مع المحللة الخلية. وتنقيته البروتينات التي ترتبط مع تسلسل ARE وتحديد الطيف الكتلي (MS). على الرغم من أن هذه الطريقة الكشف عن الجمعيات التي تحدث خارج الإعدادات الخلوية (أي في المختبر)، فإنه من المرجح أن يتم تعديل في المستقبل وذلك لتقديم الأبتامرات في الجينوم وتمكين وبالتالي عزل البروتينات المرتبطة مرنا أثناء وجوده في الوسط الخلوي (أي في الجسم الحي). في الخميرة، حيث يتم وضع التلاعب الجيني جيدا، وطريقة سريعة (وضعت في مختبر البروفيسور جيف Gerst) تقدم وجهة نظر في الجسم الحي الجمعيات 19. السريع يجمع بين MS2- محددة وملزمة من البروتين معطف MS2 قوي (CP) إلى تسلسل الحمض النووي الريبي MS2، والمجال ملزم streptavidin (متوسط ​​ضغط الدم الانقباضي) لstreptavidin الخرز مترافق. وهذا يمكن تنقية فعالة لمن mRNAs الموسومة MS2 من خلال حبات streptavidin. وعلاوة على ذلك، والتعبير من 12 نسخ من حلقات MS2 يسمح ما يصل الى ستة MS2-البرامج القطرية لربط في الوقت نفسه إلى الحمض النووي الريبي وزيادة كفاءة عزلتها. ولذلك اقترح هذا البروتوكول لتمكين تحديد البروتينات المرتبطة مرنا رواية أخرى يتعرضون عينات مزال لتحليل البروتينات التي مطياف الكتلة. نحن استخدمت مؤخرا السريع لتحديد البروتينات الجديدة المرتبطة الخميرة PMP1 مرنا 20 تم إظهار PMP1 مرنا سابقا لتترافق مع غشاء ER وعثر 3 &#39;المنطقة غير مترجمة لها (UTR) أن يكون محددا رئيسيا في هذه الجمعية 21. وهكذا، الممارسات التجارية التقييدية التي تربط PMP1 3 &#39;UTR من المرجح أن يلعب دورا هاما في توطين لها. يتبع السريع من قبل الكروماتوجرافي السائلأدى ذ الكتلة الطيفي / قياس الطيف الكتلي (LC-MS / MS) في تحديد العديد من البروتينات الجديدة التي تتفاعل مع PMP1 20. هنا، ونحن نقدم بروتوكول مفصل لمنهجية سريعة، والضوابط الهامة التي تحتاج إلى القيام به، ونصائح تقنية قد تحسين المحصول وخصوصية.

<table border="0" cellpadding="0" cellspacing="0" width="632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tris</td>
			<td style="width:107px;">sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>bio-lab</td>
			<td align="right">1981232300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol (pH 4.3)</td>
			<td>sigma</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol: Chloroform (5:1, pH 4.3)</td>
			<td>sigma</td>
			<td>P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>bio-lab</td>
			<td align="right">3080521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Frutarom</td>
			<td align="right">5551820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>sigma</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Calbiochem</td>
			<td align="right">492016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl Flouride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonuclease&#160; Inhibitor</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Beads</td>
			<td>Sartorius</td>
			<td>BBI-8541701</td>
			<td>0.4-0.6mm diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini BeadBeater</td>
			<td>BioSpec</td>
			<td colspan="2">Mini BeadBeater 16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium</td>
			<td>Sigma</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin</td>
			<td>Sigma</td>
			<td>A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads</td>
			<td>GE Healthcare&#160;</td>
			<td>17-5113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA</td>
			<td>Sigma</td>
			<td>R8508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Bacto</td>
			<td dir="LTR">288620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Bacto</td>
			<td dir="LTR">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 x Phosphate-Buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 x Laemmli Sample Buffer (LSB)</td>
			<td></td>
			<td></td>
			<td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot phenol lysis buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 M Sodium Acetate pH 5.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% and 70% Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RaPID lysis buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &#181;g/ml Leupeptin, 10 &#181;g/ml Aprotinin, 10 &#181;g/ml Soybean Trypsin Inhibitor, 10 &#181;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&#160; Inhibitor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Cross-linking reversal buffer</td>
			<td></td>
			<td></td>
			<td dir="LTR">100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RaPID wash buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris-HCl pH 7.5,&#160; 300 mM NaCl, 0.5% NP-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EDTA pH 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Stain Plus Kit</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0449</td>
			<td>For detecting proteins in polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SD selective medium&#160;</td>
			<td></td>
			<td></td>
			<td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-eEF3 (EF3A,YEF3)</td>
			<td colspan="2">Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td>
			<td>1:5,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti GFP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8334</td>
			<td>1:3,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti rabbit IgG-HRP conjugated</td>
			<td>SIGMA</td>
			<td>A9169</td>
			<td>1:10,000</td>
		</tr>
	</tbody>
</table>

novel rna-binding proteins isolation by the rapid methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

السريع تمكن من عزل الحمض النووي الريبي هدف محدد مع البروتينات المرتبطة بها. حاسمة لنجاحها والحفاظ على الحمض النووي الريبي سليمة قدر الإمكان، وبالتالي الحصول على كمية كافية من البروتينات. لتحديد كفاءة العزل ونوعية من الحمض النووي الريبي، يتم إجراء ...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

رواية عزل-ملزم RNA والبروتينات التي السريع المنهجية

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

The overall goal of this procedure is to efficiently isolate specific RNA with proteins that are associated with the RNA in vivo. This method can help answer key questions in RNA metabolism and regulation, such as what is a distinct set of fau-bee-pees bound to a particular transcript. The main advantage of this technique is the high efficiency of isolation, which allows follow up identification of noble mRNA associated proteins.The rapid methodology utilizes a yeast strain that co-expresses MS2-tagged MRNA and a fusion protein of an MS2 binding protein and a streptavadin binding protein. Grow 500 milliliters of yeast cells at 30 degrees Celsius in synthetic dextrose or SD selective medium to an OD600 of 0.8 to 1.0. When the cells are ready, centrifuge at 3000 times G for 4 minutes at room temperature and discard the supernatant.Wash the cells in PBS and centrifuge again. Discard the supernatant and re-suspend the cells in an equal volume of SD without methionine. Incubate for 45 to 60 minutes at 30 degrees celcius to induce expression of the fusion protein.After 45 to 60 minutes, set aside 10 milliliters of the cells for RNA extraction and use the remaining cells for rapid purification. To begin this procedure, add to the cells 1.35 milliliters of 37%formaldehyde, to a final concentration of 0.1%to cross-link the RNA-protein complexes. Continue incubating at 30 degrees Celsius for 10 minutes.To stop cross-linking, add 26.5 milliliters of 2.5 molar glycine, to a final concentration of 0.125 molar, and incubate at 30 degrees Celsius for 3 minutes. From here on, keep everything on ice and work quickly to minimize RNA degradation. Centrifuge the cells at 3000 times G for 4 minutes at 4 degrees Celsius.Discard the supernatant, add 45 milliliters of cold PBS, and centrifuge again. Remove the supernatant and re-suspend the cells in 5 milliliters of rapid lysis buffer that contains a high concentration of Rnase inhibitors. Divide the cell suspension into aliquots of 500 microliters in screw-capped microfuge tubes and add approximately 400 milligrams of chilled glass beads to each aliquot.Lyse the cells in a bead-beater for three minutes. Immediately put the lysate on ice. Transfer the lysate to new microfuge tubes.Cut the top off a 5 milliliter syringe and put it on an empty 15 milliliter tube to serve as an adapter for screw-cap tubes. Use a hot needle to pierce a small hole in the bottom of each tube and place them on top of the adapted 15 milliliter tubes. Centrifuge the tub assemblies at 3000 times G for one minute at 4 degrees Celsius to elute the lysate to the 15 milliliter tubes.Transfer the flowthrough from each 15 milliliter tube to a new 1.5 milliliter tube and centrifuge at 10, 000 times G for 10 minutes at 4 degrees Celsius to clear the cell debris. Pool the supernatant from all 1.5 milliliter tubes into a single 15 milliliter tube. Set aside 1/50th and 1/100th volume of the lysate for RNA and protein isolation respectively.Begin the rapid procedure for isolating RNA protein complexes by adding 300 micrograms of Avidin to the cell lysate. Incubate for 30 minutes at 4 degrees Celsius under constant rolling. While the cell lysate is being incubated with Avidin, pre-wash the streptavidin beads, transfer about 300 microliters of the streptavadin bead slurry to weigh 1.5 milliliters microcentriuge tube, centrifuge the preparation, and then remove the upper supernatant which will result in 250 microliters of beads.Add 1 milliliter of rapid lysis buffer to the beads and centrifuge at 660 times G for 2 minutes at 4 degrees Celsius. Remove the supernatant and wash again with rapid lysis buffer. To block the beads, add 0.5 milliliters of rapid lysis buffer, 0.5 milliliters of BSA, and 10 microliters of yeast TRNA and incubate for 1 hour at 4 degrees Celsius with constant rotation.Wash the beads twice with 1 milliliter of rapid lysis buffer. Add 250 microliters of the pre-washed streptavidin beads to the avidin containing lysate and incubate for 1 hour at 4 degrees Celsius with constant rotation. This incubation time is a compromise between providing sufficient binding time and minimizing the chances for RNA degradation.After one hour, centrifuge at 660 times G for 2 minutes at 4 degrees Celsius to clear the streptavidin beads. Transfer the supernatant to a 15 milliliter tube. Set aside 1/50th and 1/100th volume of the lysate for RNA and protein isolation respectively.Add 1 milliliter of rapid lysis buffer to the beads. Mix by pipetting, transfer to a new 1.5 milliliter microcentrifuge tube and centrifuge at 660 times G for 2 minutes at 4 degrees Celsius. Wash three more times with rapid lysis buffer.After the final wash with rapid lysis buffer, remove the supernatant, add 1 milliliter of rapid wash buffer, and rotate for 5 minutes at 4 degrees Celsius. Centrifuge and wash 2 more times with rapid wash buffer. Wash the beads with 1 milliliter of PBS and centrifuge as before.Remove the supernatant, add to the beads 120 microliters of PBS, and rotate for 5 minutes in a roller. Centrifuge as before and collect all the supernatant for RNA and protein extraction as the wash sample. To elute RNA and RNA-associated proteins, add 120 microliters of 6 millimolar biotin in PBS to the beads, and incubate for 45 minutes at 4 degrees Celsius with constant rotation.Centrifuge the beads and transfer the eluded material to a new 1.5 milliliter tube. Spin the eluted sample again and transfer the upper phase to a new 1.5 milliliter tube to ensure no carry-over of beads. Take samples for RNA or protein extraction.To determine the isolation efficiency and quality of RNA, Northern Analysis was performed for two tagged RNAs. PMP1 and FPR1, from cast cell lysis, rapid cell lysis, and after elution with biotin. Ribosomal RNAs were detected by ethidium bromide staining, and the lack of ribosomal RNAs in the elution sample indicates the stringency of purification.The stringency and specificity of the purification are further demonstrated by the lack of a signal of an untagged MRNA in the elution samples. The apparent signal in the FPR1 lane, which is not of the size of ACT1, is a leftover from previous hybridization with the MS2L probe. Although the input RNA is somewhat more degraded compared to the RNA that was purified by the Hot Phenol protocol, a significant amount of full length tagged RNA is isolated specifically, as revealed by the strong signal in the elution fractions.Protein samples can be analyzed by SDS-PAGE and silverstaining prior to proteomics analysis. The MS2-CP-GFP-SBP fusion protein, indicated by the arrow, demonstrates equal protein loading. The asterisks indicate bands with differential intensities that were later cut out of the gel for mass-spectrometry analysis.While attempting this procedure, it's important to wear gloves, ensure a clean work-area, prepare solution with RNA free water, and work quickly and efficiently to reduce protocol time. Don't forget that working with reagents like phenol and formaldehyde can be extremely hazardous, and precautions such as working in a chemical hood should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to efficiently and effectively purify RNA of fayn-tress and its associated body.Following this procedure, other methods like RNA-Seq can be performed in order to detect RNAs that bind the transcript of interest.

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

اقترانية التزاوج فحوصات لتحليل تسلسل محدد من البروتينات نقل تشارك في الإقتران البكتيرية

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

حركية توليف الحمض النووي متخلفة حبلا<em&gt; في المختبر</em&gt; من البروتينات النسخ المتماثل الجراثيم T7

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

موقع التكامل ثنائي الاتجاه للفيروسات القهقرية منهجية ير والتحليل الكمي للبيانات سير العمل

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

منهجية موحدة لدراسة المحدثة للطفرات في الموقع كدالة لإصلاح نقطة تحور تحفزها كريسبر/Cas9 وسودن في الخلايا البشرية

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

منهجية للكشف عن دقة مثلايشن الحمض النووي

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Novel Sequence Discovery by Subtractive Genomics

اكتشاف رواية تسلسل الجينوم مطروح

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

تسلسل mRNA من الدم الكامل باستخدام التسلسل نانو

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

تحليلات البروتيوميات الجماعي القائم على قياس الطيف الكتلي باستخدام قاعدة البيانات أوبينبروت لكشف النقاب عن البروتينات رواية مترجمة من إطارات القراءة المفتوحة غير متعارف عليه

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

اختبار لتحديد كمية البروتين-RNA الربط في البكتيريا

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

استكشاف مساحة التسلسل لتحديد مواقع الربط للبروتينات الملزمة بالحمض النووي الريبي التنظيمية

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...