Name: rapid and cost-effective rna extraction of rat pancreatic tissue
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وقد حظيت هذه الدراسة بدعم مالي من جامعة شيراز للعلوم الطبية (منحة رقم 93-01-01-7178\03-07-2014). نشكر السيد زوموروديان والسيد رستمي في قسم التعليم الإلكتروني في العلوم الطبية، والمدرسة الافتراضية ومركز التميز في التعليم الإلكتروني، جامعة شيراز للعلوم الطبية لتحرير الفيديو.

تم الحصول على الموافقة الأخلاقية لهذه الدراسة من جامعة شيراز للعلوم الطبية (رقم الموافقة: 93-01-01-7178\03-07-2014).
ملاحظة: استخدام الذكور Sprague-Dawley الفئران وزنها 250 غرام. وضع القارورة التي تحتوي على شظية من أنسجة البنكرياس مغمورة في الحمض النووي الريبي استقرار الكاشف في خزان ...

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S., Aubin, R. A., Bura, C. A., Curran, I. H., Matula, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+recovery+of+intact+RNA+from+rat+pancreas.">Ensuring recovery of intact RNA from rat pancreas.</a> Molecular Biotechnology. 6 (3), 359-362 (1996).</li><li>Hernandez, G. E., Mondala, T. S., Head, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+a+novel+room+temperature+RNA+storage+medium+for+compatibility+in+microarray+gene+expression+analysis.">Assessing a novel room temperature RNA storage medium for compatibility in microarray gene expression analysis.</a> Biotechniques. 47 (2), 667 (2009).</li><li>Mullin, A. E., Soukatcheva, G., Verchere, C. B., Chantler, J. 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R., Sambrook, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+win+the+battle+with+RNase.">How to win the battle with RNase.</a> Cold Spring Harbor Protocols. (2), 101857 (2019).</li><li>Li, D. -. S., Yuan, Y. -. H., Tu, H. -. J., Dai, L. -. j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">A protocol for islet isolation from mouse pancreas.</a> Nature Protocols. 4 (11), 1649 (2009).</li><li>Armstrong, J. A., Schulz, J. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis.">Agarose gel electrophoresis.</a> Current Protocol: Essential Laboratory Techniques. (1), 1-20 (2008).</li><li>Aranda, P. S., LaJoie, D. 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F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning:+a+laboratory+manual.">Molecular cloning: a laboratory manual.</a> Cold spring harbor laboratory press. , (1989).</li><li>Potenza, N., 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=Hybridase+activity+of+human+ribonuclease-1+revealed+by+a+real-time+fluorometric+assay.">Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay.</a> Nucleic Acids Research. 34 (10), 2906-2913 (2006).</li><li>Jackson, D., Lewis, F., Taylor, G., Boylston, A., Quirke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+extraction+of+DNA+and+RNA+and+analysis+by+the+polymerase+chain+reaction.">Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.</a> Journal of Clinical Pathology. 43 (6), 499-504 (1990).</li><li>Quesada, I., Tudur&#237;, E., Ripoll, C., Nadal, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+the+pancreatic+&#945;-cell+and+glucagon+secretion:+role+in+glucose+homeostasis+and+diabetes.">Physiology of the pancreatic &#945;-cell and glucagon secretion: role in glucose homeostasis and diabetes.</a> Journal of Endocrinology. 199 (1), 5-19 (2008).</li><li>Quertinmont, E., Nicaise, C., Gustot, T., Deviere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Homogenization+with+the+MagNA+Lyser+Instrument+for+Total+RNA+Extraction+Using+the+TriPure+Reagent.">Tissue Homogenization with the MagNA Lyser Instrument for Total RNA Extraction Using the TriPure Reagent.</a> Liver (mg). 100 (100), 100 (2004).</li><li>Dastgheib, S., Irajie, C., Assaei, R., Koohpeima, F., Mokarram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+RNA+extraction+from+rat+pancreatic+tissue.">Optimization of RNA extraction from rat pancreatic tissue.</a> Iranian Journal of Medical Sciences. 39 (3), 282 (2014).</li></ol>

في البيولوجيا الجزيئية من المهم الحصول على RNA عالية الجودة. وجود الإنزيمات ريبونوكليز في الخلايا والأنسجة سرعان ما يتحلل الجيش الملكي النيبالي ويجعل عملية الاستخراج معقدة. RNases هي الإنزيمات مستقرة تعمل دون أي عوامل. كميات صغيرة من RNase كافية لتدمير الجيش الملكي النيبالي. عندما تتم إزالة أنس...

يمكن نسخ البيانات الجينية الهيكلية إلى منتج وظيفي من خلال التعبير الجيني. يستخدم تحليل الحمض النووي الريبي لاكتشاف الاختلافات في التعبير الجيني عبر ظروف مختلفة. هناك عدد من الطرق لاستخراج الأحماض النووية على النحو التالي: الثيوسيانات guanidinium، والاستخراج عن طريق الفينول- الكلوروفورم، والكرومات القائم على السليلوز، واستخراج بواسطة مصفوفات السيليكا، وتبادل الأنيون1،2.
ويتأثر الكشف السليم عن التعبير الجيني بسلامة الحمض النووي الريبي المعزول عن الأنسجة؛ ولذلك، فمن الأهمية بمكان لتقييم سلامة الحمض النووي الريبي معزولة عن الأنسجة قبل إجراء مزيد من الاختبارات لأن الاختبارات الجزيئية التكميلية على RNA منخفضة الجودة قد تعرض للخطر نتائج التطبيق التشخيصي. وهكذا، هناك حاجة إلى RNA سلامة عالية للاختبارات البيولوجية الجزيئية مع تطبيقات التشخيص المختلفة: كمية RT-PCR، صفائف صغيرة، مقايسة حماية ribonuclease، تحليل البقعة الشمالية، رسم خرائط الحمض النووي الريبي، وبناء مكتبة cDNA3،4.
RNA يصبح غير مستقر نوعا ما بعد أن يتم الاحتفاظ بها لفترة طويلة. شظايا مرنا طويلة أكثر من 10 كيلو بايت عرضة بشكل خاص للتدهور5،6. وبالتالي، يجب على الباحثين النظر في مختلف العوامل التي تؤثر على سلامة الحمض النووي الريبي المنقى. يجب حماية نقاء الحمض النووي الريبي ضد RNases والبروتينات والحمض النووي الجينومي والتلوث المثبط الأنزيمي. بالإضافة إلى ذلك، يجب أن تكون نسبة امتصاص أفضل ومقبولة من الحمض النووي الريبي إلى الأشعة فوق البنفسجية (260/280) ضمن نطاق 1.8-2.0 مع الحد الأدنى من التشظي على الكهرباء. وقد مكنت التقنيات المختبرية التي وضعت مؤخرا العلماء لتقييم سلامة عينة التحليل الجزيئي أكثر عمليا7،8.
ومن الصعب استخراج الحمض النووي الريبي غير التالفة من أنسجة البنكرياس من أنواع أخرى من الأنسجة بسبب كمية عالية من ريبونوكليس (RNases). ومع ذلك ، فإن طرق الاستخراج الحالية ، وهي القذف السريع للأنسجة البنكرياسية من تجويف البطن والتجانس في درجات حرارة منخفضة لإعاقة RNases ، أثبتت أنها غير فعالة7،8،9،10،11،12،13،14.
والغرض من هذه الدراسة التجريبية المقارنة هو تعديل ومقارنة الأساليب القائمة لتحديد أكثر الطرق كفاءة. وتحقيقا لتلك الغاية، تم تعديل بروتوكولات مختلفة لاستخراج الحمض النووي الريبي ومقارنتها. وكان الهدف منها على وجه التحديد هو تحديد الطريقة الأقل تكلفة التي تتطلب الحد الأدنى من أنسجة البنكرياس.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Merck</td>
			<td>116801</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Atoclave</td>
			<td>Teb Zaim</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>107024</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonate (DEPC)-treated water</td>
			<td>Sigma</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>sigma</td>
			<td>60-00-4</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Electrophoresis tank</td>
			<td>Payapajoohesh</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Eppendorf microTube</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>EtBr</td>
			<td>sigma</td>
			<td>E 8751</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>81870</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Falcon Tube</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck</td>
			<td>344198</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Merck</td>
			<td>344206</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Homogenizer-sunicator</td>
			<td>Microson XL 2000</td>
			<td></td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>sigma</td>
			<td>19516</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>sigma</td>
			<td>1867-66-9</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Jal Tajhiz</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>Mgnetic stirrer</td>
			<td>Labrotechnik</td>
			<td></td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Micropipette Tips</td>
			<td>Extragene</td>
			<td></td>
			<td>Taiwan</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>sigma</td>
			<td>85022106</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Na AC</td>
			<td>Merck</td>
			<td>567422</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>109137</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Teb Zaim</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Knick</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>RNA Later/RNA stabilization reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Surgical instrument</td>
			<td>Agn Thos</td>
			<td></td>
			<td>German made</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>AvaPezeshk</td>
			<td></td>
			<td>Iran</td>
		</tr>
		<tr>
			<td>TriPure reagent/RNA extraction reagent</td>
			<td>Roche</td>
			<td>11667157001</td>
			<td>USA</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Labinco</td>
			<td></td>
			<td>Netherland</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>zylazine</td>
			<td>sigma</td>
			<td>7361-61-7</td>
			<td>Germany</td>
		</tr>
	</tbody>
</table>

rapid and cost-effective rna extraction of rat pancreatic tissue

بغض النظر عن طريقة الاستخراج، يتم استخراج الحمض النووي الريبي الأمثل من الأنسجة وخطوط الخلايا في أربع مراحل: 1) التجانس، 2) denaturation فعالة من البروتينات من الجيش الملكي النيبالي، 3) ريبونوكيليشنيشن، و 4) إزالة التلوث من الحمض النووي، والبروتينات، والكربوهيدرات. ومع ذلك، فمن شاق جدا للحفاظ على سلامة الجيش الملكي النيبالي عندما تكون هناك مستويات عالية من RNase في الأنسجة. التلقائي التلقائي يجعل من الصعب جداً استخراج الحمض النووي الريبي من أنسجة البنكرياس دون الإضرار به. وهكذا، هناك حاجة إلى طريقة عملية استخراج الحمض النووي الريبي للحفاظ على سلامة أنسجة البنكرياس أثناء عملية الاستخراج. أجريت دراسة تجريبية ومقارنة للبروتوكولات الموجودة عن طريق الحصول على 20-30 ملغ من أنسجة البنكرياس الفئران في أقل من 2 دقيقة واستخراج RNA. تم تقييم النتائج بواسطة الكهرباء. وأجريت التجارب ثلاث مرات لتعميم النتائج. غمر أنسجة البنكرياس في مُستفد تثبيت الحمض النووي الريبي في -80 درجة مئوية لـ 24 ساعة أثمرت RNA عالية النزاهة، عندما تم استخدام كاشف استخراج الحمض النووي الريبي كمكاد للكاشف. وكانت النتائج التي تم الحصول عليها مماثلة للنتائج التي تم الحصول عليها من مجموعات المواد التجارية مع ربط عمود الدوران.

تقييم سلامة الجيش الملكي النيبالي في مُكشف استخراج الحمض النووي الريبي وفقا لبروتوكول روتيني وتعديلي جراحية دون كاشف تثبيت الحمض النووي الريبي لوحظت عصابات غير مقبولة بعد استخراج الحمض النووي الريبي مع كاشف استخراج الحمض النووي الريبي من بروتوكول جراحية روتينية. لين 1 يظهر ال...

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Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

نقاء وسلامة الجيش الملكي النيبالي المعزول هو خطوة حيوية في اختبار الحمض النووي الريبي تعتمد. هنا، نقدم طريقة عملية وسريعة وغير مكلفة لاستخراج الحمض النووي الريبي من كمية صغيرة من أنسجة البنكرياس غير التالفة.

استخراج RNA سريع وفعال من حيث التكلفة من أنسجة البنكرياس الجرذ

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

مرحباً بالجميع لقد صممنا, مباشرة, طريقة وصفية سريعة في فئة لاستخراج الحمض النووي الريبي كفاءة من أنسجة البنكرياس. الميزة الرئيسية لهذه الطريقة هو إجراء الأمثل لاستخراج RNA من الحد الأدنى من كمية الأنسجة بسبب الانحلال التلقائي. مرحبا مرة أخرى. سيتم إجراء التظاهر من قبل ساناز Dastghaib، وهذا هو الطالب بلدي دكتوراه في مختبرنا في قسم الكيمياء الحيوية في جامعة شيراز للعلوم الطبية.أولا إعداد مكان الجراحة، ووضع جميع المتطلبات تحت غطاء محرك السيارة. إلى تعطيل RNase تعقيم جميع أدوات الجراحة في الفرن. حقن الكيتامين-زيلازين intraperitoneally 80/8 مليغرام لكل كيلوغرام. إضافة 1 cc.RNA مُثبّت تثبيت في microtube المناسبة لتسكن تنشيط endonuclease في الأنسجة الموجودة. ضع الفأر على الفور على اللوحة الجراحية. رئيس بعيدا الجراح لشق خط الوسط. ثم تعقيم كل سطح البطن مع 70٪ من الكحول لإزالة شعر الفئران. قطع الجلد لعملية جراحية. تم إجراء شق إعادة تشكيل لفتح البطن من منطقة العانة إلى الساقين الأماميتين. اقلب أعضاء البطن إلى الجانب الأيسر، لفضح البنكرياس. العثور بعناية على أنسجة البنكرياس، والتي تنتشر في تجويف البطن. حدد مكان المنطقة المعروضة لمعرفة البنكرياس دون الخلط بينه وبين الأنسجة الدهنية. إزالة البنكرياس على الفور 20 إلى 30 ملليغرام من تجويف البطن في أقل من دقيقتين. وضع الأنسجة إزالتها في أنبوب صغير بسرعة كبيرة وقطع عليه مع القاطع استريد لاختراق كاشف تثبيت الحمض النووي الريبي لفصل الأنسجة. وضع microtube في خزان النيتروجين لتجميد على الفور. وينبغي أن تبقى جميع الأنابيب الخاصة بهم microTube في 80

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Research

JoVE Journal

Biochemistry

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

بروتوكول محسن لاستخراج البروتينات من صمام ميترال الإنسان

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

جمع الأنسجة الدهنية للماوس وتجهيزها لتحليل الحمض النووي الريبي

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

تحليل توبرينتينج "الترجمة بدء تشكيل معقدة" في مرناس الثدييات

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

عزل ميتوريبوسومي من الخلايا HEK السريع

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

التقاط وتحديد البروتينات الحمض النووي الريبي ملزم باستخدام نقرة الكيمياء-وساعدت الجيش الملكي النيبالي-إينتيراكتومي التقاط استراتيجية (كاريك)

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Extraction and Analysis of Taiwanese Green Propolis

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

استخراج وتحليل للتايوانيين دنج الأخضر

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

دراسة التمثيلات الحمض النووي الريبي للبروتين كيناز المنشط الجيش الملكي النيبالي خلال دورة الخلية الثديية

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

سريع للغاية ومحددة وضع العلامات الأيضية من الحمض النووي الريبي في فيفو مع 4-ثيوراسيل (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

استخراج جانجليوسيد وتنقيته وتوصيفه

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.