Name: rapid and cost-effective rna extraction of rat pancreatic tissue
Uploaded: 2020-09-19T14:00:00
Description: Watch this Scientific Journal Video about Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue at JoVE.com

Den föreliggande studien stöddes ekonomiskt av Shiraz University of Medical Sciences (anslag nr 93-01-01-7178\03-07-2014). Vi tackar Mr Zomorodian och Mr Rostami vid institutionen för e-lärande i medicinska vetenskaper, Virtual School och Center of Excellence i e-Learning, Shiraz University of Medical Sciences för redigering av video.

Etiskt godkännande för denna studie erhölls från Shiraz University of Medical Sciences (Godkännandenummer: 93-01-01-7178\03-07-2014). approval
OBS: Använd handjur Sprague–Dawley råttor som väger 250 g. Placera injektionsflaskan som innehåller en flisa av bukspottskörtelns vävnad nedsänkt i RNA-stabiliserande reagens i en tank för flytande kväve vid -80 °C och använd RNA-utvinningsreagenslösning för att upprätthålla RNA:s integritet.
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<ol>
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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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+situ+ductal+perfusion+to+facilitate+isolation+of+high-quality+RNA+from+mouse+pancreas.">Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.</a> Biotechniques. 40 (5), 617 (2006).</li><li>Li, 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=A+modified+method+using+TRIzol+reagent+and+liquid+nitrogen+produces+high-quality+RNA+from+rat+pancreas.">A modified method using TRIzol reagent and liquid nitrogen produces high-quality RNA from rat pancreas.</a> Applied Biochemistry and Biotechnology. 158 (2), 253-261 (2009).</li><li>Griffin, M., Abu-El-Haija, M., Abu-El-Haija, M., Rokhlina, T., Uc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+and+versatile+method+for+obtaining+high+quality+rna+from+pancreas.">A simplified and versatile method for obtaining high quality rna from pancreas.</a> Biotechniques. 52 (5), 332 (2012).</li><li>Jun, 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=Method+optimization+for+extracting+high-quality+RNA+from+the+human+pancreas+tissue.">Method optimization for extracting high-quality RNA from the human pancreas tissue.</a> Translation Oncology. 11 (3), 800-807 (2018).</li><li>Green, M. 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. M., Jorcyk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+gel:+a+simple+agarose+gel+for+analyzing+RNA+quality.">Bleach gel: a simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li><li>Sambrook, J., Fritsch, E. 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>

Inom molekylärbiologi är det livsviktigt att få RNA av hög kvalitet. Förekomsten av ribonukleasenzymerna i celler och vävnader försämrar snabbt RNA och gör extraktionen komplex. RNaser är stabila enzymer fungerar utan några co-faktorer. Små mängder av RNase är tillräckliga för att förstöra RNA. När råttpanspottskörtelns vävnad avlägsnas från bukhålan är det nödvändigt att desinficera de kirurgiska instrumenten genom starka rengöringsmedel, skölj dem noggrant och lägg dem i en ugn i minst 4 ...

Strukturella gendata kan transkriberas till en funktionell produkt genom genuttryck. RNA-analys används för att upptäcka skillnader i genuttryck över olika förhållanden. Det finns ett antal metoder för att extrahera nukleinsyror enligt följande: guanidiniumtiocyanat, extraktion via fenol-kloroform, cellulosabaserad kromatografi, extraktion genom kiseldioxidmatriser, och anjon-utbyte1,2.
Korrekt detektion av genuttryck påverkas av integriteten hos RNA som isolerats från vävnader; därför är det livsviktigt att utvärdera integriteten hos RNA som isolerats från vävnader innan ytterligare tester genomförs eftersom kompletterande molekylära tester på RNA av låg kvalitet kan äventyra diagnostiska applikationsresultat. Således är hög integritet RNA behövs för molekylärbiologiska tester med olika diagnostiska tillämpningar: kvantitativa RT-PCR, mikro-arrayer, ribonukleas skydd assay, norra blot analys, RNA-mappning, och cDNA bibliotekkonstruktion 3,4.
RNA blir ganska instabilt efter att ha hållits länge. Långa mRNA-fragment över 10 kb är särskilt känsliga för nedbrytning5,6. Således måste forskare överväga olika faktorer som påverkar integriteten hos renat RNA. Renheten hos RNA måste skyddas mot RNaser, proteiner, genomiskt DNA, och enzymatisk hämmar kontaminering. Dessutom måste det bästa och acceptabla absorptionsförhållandet för RNA till UV (260/280) ligga inom intervallet 1,8-2,0 med minsta fragmentering över elektrofores. Nyligen utvecklade laboratorietekniker har gjort det möjligt för forskare att utvärdera integriteten hos molekylär analysprov mer praktiskt7,8.
Det är mycket svårare att extrahera oskadade RNA från bukspottskörteln vävnad än andra typer av vävnader på grund av den höga mängden av ribonukleases (RNases). Dock har befintliga extraktionsmetoder, nämligen den snabba utskjutningen av bukspottskörteln vävnad från bukhålan och homogenisering vid låga temperaturer för att hindra RNases, visat sigvara ineffektiva 7,8,9,10,11,12,13,14.
Syftet med den föreliggande jämförande experimentella studien är att modifiera och jämföra befintliga metoder för att bestämma de mest effektiva metoderna. För detta ändamål ändrades och jämfördes olika protokoll för RNA-utvinning. Det var särskilt syftar till att fastställa den billigaste metoden kräver en minsta mängd bukspottskörteln vävnad.

<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

Oavsett utvinningsmetoden, optimerad RNA utvinning av vävnader och cellinjer utförs i fyra steg: 1) homogenisering, 2) effektiv denaturering av proteiner från RNA, 3) ribonukleas inaktivering, och 4) avlägsnande av kontaminering från DNA, proteiner, och kolhydrater. Det är dock mycket mödosamt att upprätthålla integriteten hos RNA när det finns höga nivåer av RNase i vävnaden. Spontan autolys gör det mycket svårt att extrahera RNA från bukspottskörteln vävnad utan att skada den. Således behövs en praktisk RNA-extraktionsmetod för att upprätthålla integriteten hos bukspottskörtelvävnader under extraktionsprocessen. En experimentell och jämförande studie av befintliga protokoll genomfördes genom att erhålla 20-30 mg av råtta bukspottskörteln vävnader på mindre än 2 minuter och extrahera RNA. Resultaten bedömdes med elektrofores. Experimenten utfördes tre gånger för generalisering av resultaten. Nedsänkning bukspottskörteln vävnad i RNA stabilisering reagens vid -80 °C för 24 h gav hög integritet RNA, när RNA extraktion reagens användes som reagens. De erhållna resultaten var jämförbara med de resultat som erhållits från kommersiella kit med spinnkolumnbindningar.

Utvärdering av RNA:s integritet i RNA-utvinningsreagensen enligt ett rutinmässigt och modifierat kirurgiskt protokoll utan RNA-stabiliseringsreagens Oacceptabla band observerades efter extraktionen av RNA med RNA-extraktionsreagensen från ett rutinmässigt kirurgiskt protokoll. Lane 1 visar RNA från levern som en kontroll. Lane 2 visar den försämrade statusen för 28S/18S rRNA-band i totalt RNA som erhållits från ett rutinmässigt kirurgiskt protokoll. När mängden bukspottskörteln vävnad m...

Watch this Scientific Journal Video about Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue at JoVE.com

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Renhet och integritet av den isolerade RNA är ett viktigt steg i RNA beroende analyser. Här presenterar vi en praktisk, snabb och billig metod för att extrahera RNA från en liten mängd oskadad bukspottskörtelvävnad.

Snabb och kostnadseffektiv RNA-utvinning av råtta bukspottskörteln vävnad

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

Hej allihopa. Vi har utformat, en okomplicerad, snabb i klassen beskrivande metod för effektiv RNA-utvinning från bukspottskörteln vävnad. Den största fördelen med denna metod är ett optimerat förfarande för att extrahera RNA från minsta mängd vävnad på grund av autolys. Hej igen. Demonstrera av förfarandet kommer att ske av Sanaz Dastghaib, det är min doktorand i vårt labb på Biochemistry Institutionen för Shiraz University of Medical Sciences.First förbereda kirurgi plats, sätta alla krav under huven. För att inaktivera RNase sterilisera alla kirurgi instrument i en ugn. Injicera ketamin-xylazin intraperitoneally 80/8 milligram per kilogram. Lägg 1 cc.RNA stabilisering reagens i korrekt microtube att bebo aktivering av enonuclease i fanns vävnader. Placera omedelbart råttan på operationsbrädan. Bege dig bort kirurgen för ett mittlinjeansion. Sedan, sterilisera alla bukytan med 70%alkohol för att ta bort råttan hår. Skär huden för operationen. Omforma snitt gjordes för att öppna buken från blygdområdet till frambenen. Vänd bukorganen till vänster sida, för att exponera bukspottkörteln. Noggrant hitta bukspottkörteln vävnad, som sprids i bukhålan. Lokalisera området under display för att ta reda på bukspottkörteln utan att förväxlas med fettvävnaden. Omedelbart ta bort 20 till 30 milligram bukspottkörtel från bukhålan på mindre än två minuter. Lägg den borttagna vävnaden i mikroröret mycket snabbt och skär den med astrideskäraren för att penetrera RNA-stabiliseringsreagensen till separerade vävnader. Placera mikroröret i kvävetank för att frysa omedelbart. Alla deras mikrorör ska hållas i de 80

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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.

Optimerat protokoll för extraktion av proteiner från humanmitralventilen

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.

Mus fettvävnad insamling och bearbetning för RNA analys

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.

Toeprinting analys av översättning inledande komplexa bildande på däggdjur mRNA

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.

Snabb isolering av Mitoribosome från HEK celler

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.

Fånga och identifiering av RNA-bindande proteiner med hjälp av klick kemi-assisted RNA-interactome Capture (CARIC) strategi

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.

Extraktion och analys av taiwanesiska grön Propolis

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.

Studera RNA interactmedlemmar av proteinkinas RNA-aktiverad under däggdjur cellcykeln

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.

Extremt snabb och specifik metabolisk märkning av RNA in vivo med 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 ...

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.

Utvinning, rening och profilering av gangliosid

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.