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

Il presente studio è stato sostenuto finanziariamente dalla Shiraz University of Medical Sciences (Grant No. Ringraziamo il Sig. Eomorodian e il Sig. Rostami presso il Dipartimento di e-Learning in Scienze Mediche, Scuola Virtuale e Centro di Eccellenza in e-Learning, Università di Scienze Mediche di Shiraz per la modifica del video.

L'approvazione etica per questo studio è stata ottenuta dalla Shiraz University of Medical Sciences (numero di approvazione: 93-01-01-7178-03-07-2014).
NOTA: Utilizzare ratti maschi Sprague-Dawley del peso di 250 g. Posizionare la fiala contenente una scheggia di tessuto pancreatico immerso nel reagente stabilizzante dell'RNA in un serbatoio di azoto liquido a -80 gradi centigradi e utilizzare la soluzione del reagente di estrazione dell'RNA per mantenere l'integrità dell'R...

<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>

Nella biologia molecolare è fondamentale ottenere RNA di alta qualità. La presenza degli enzimi ribonucleasi nelle cellule e nei tessuti degrada rapidamente l'RNA e rende l'estrazione complessa. Le rule sono enzimi stabili che funzionano senza co-fattori. Piccole quantità di RNase sono adeguate per distruggere l'RNA. Quando il tessuto pancreatico del ratto viene rimosso dalla cavità addominale, è necessario disinfettare gli strumenti chirurgici con detergenti forti, sciacquarli accuratamente e metterli in forno per ...

I dati genetici strutturali possono essere trascritti in un prodotto funzionale attraverso l'espressione genica. L'analisi dell'RNA viene utilizzata per scoprire le differenze nell'espressione genica in diverse condizioni. Ci sono una serie di metodi per estrarre gli acidi nucleici come segue: guanidinium thiocyanate, estrazione tramite fenolo-cloroformio, cromatografia a base di cellulosa, estrazione da matrici di silice e scambio dianioni 1,2.
La corretta rilevazione dell'espressione genica è influenzata dall'integrità dell'RNA isolato dai tessuti; pertanto, è fondamentale valutare l'integrità dell'RNA isolato dai tessuti prima che vengono effettuati ulteriori test perché test molecolari complementari su RNA di bassa qualità possono compromettere i risultati dell'applicazione diagnostica. Pertanto, l'RNA ad alta integrità è necessario per i test biologici molecolari con diverse applicazioni diagnostiche: RT-PCR quantitativo, micro-array, analisi della protezione della ribonucleasi, analisi delle macchie settentrionali, mappatura dell'RNA e costruzione della libreria cDNA3,4.
L'RNA diventa piuttosto instabile dopo essere stato tenuto a lungo. Frammenti di mRNA lunghi oltre 10 kb sono particolarmente suscettibili alla degradazione5,6. Pertanto, i ricercatori devono considerare vari fattori che influenzano l'integrità dell'RNA purificato. La purezza dell'RNA deve essere protetta da RNases, proteine, DNA genomico e contaminazione da inibitori enzimatici. Inoltre, il miglior e accettabile rapporto di assorbimento tra RNA e UV (260/280) deve essere compreso nell'intervallo di 1,8-2,0 con frammentazione minima sull'elettroforesi. Tecniche di laboratorio sviluppate di recente hanno permesso agli scienziati di valutare l'integrità del campione di analisi molecolarepiù praticamente 7,8.
È molto più difficile estrarre l'RNA non danneggiato dal tessuto pancreatico rispetto ad altri tipi di tessuti a causa dell'elevata quantità di ribonucleasi (RNases). Tuttavia, i metodi di estrazione esistenti, vale a dire la rapida espulsione del tessuto pancreatico dalla cavità addominale e l'omogeneizzazione a basse temperature per ostacolare le RNases, si sono dimostrati inefficaci7,8,9,10,11,12,13,14.
Lo scopo dell'attuale studio sperimentale comparativo è quello di modificare e confrontare i metodi esistenti per determinare i metodi più efficienti. A tal fine, vari protocolli di estrazione dell'RNA sono stati modificati e confrontati. Era specificamente finalizzato a determinare il metodo meno costoso che richiede una quantità minima di tessuto pancreatico.

<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

Indipendentemente dal metodo di estrazione, l'estrazione ottimizzata dell'RNA dei tessuti e delle linee cellulari viene effettuata in quattro fasi: 1) omogeneizzazione, 2) denaturazione efficace delle proteine da RNA, 3) inattivazione della ribonuclea e 4) rimozione della contaminazione da DNA, proteine e carboidrati. Tuttavia, è molto laborioso mantenere l'integrità dell'RNA quando ci sono alti livelli di RNase nel tessuto. L'autolisi spontanea rende molto difficile estrarre l'RNA dal tessuto pancreatico senza danneggiarlo. Pertanto, è necessario un metodo pratico di estrazione dell'RNA per mantenere l'integrità dei tessuti pancreatici durante il processo di estrazione. Uno studio sperimentale e comparativo dei protocolli esistenti è stato effettuato ottenendo 20-30 mg di tessuti pancreatici di ratto in meno di 2 minuti ed estraendo l'RNA. I risultati sono stati valutati per elettroforesi. Gli esperimenti sono stati effettuati tre volte per la generalizzazione dei risultati. Immergere il tessuto pancreatico nel reagente di stabilizzazione dell'RNA a -80 gradi centigradi per 24 h ha prodotto RNA ad alta integrità, quando il reagente di estrazione dell'RNA è stato utilizzato come reagente. I risultati ottenuti sono stati paragonabili ai risultati ottenuti da kit commerciali con attacchi di colonna di spin.

Valutazione dell'integrità dell'RNA nel reagente di estrazione dell'RNA secondo un protocollo chirurgico di routine e modificato senza reagente di stabilizzazione dell'RNA Dopo l'estrazione dell'RNA sono state osservate bande inaccettabili con il reagente di estrazione dell'RNA da un protocollo chirurgico di routine. La corsia 1 mostra l'RNA dal fegato come controllo. La corsia 2 mostra lo stato degradato delle bande di rRNA 28S/18S nell'RNA totale ottenuto da un protocollo chirurgico di routine. Qua...

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

La purezza e l'integrità dell'RNA isolato è un passo vitale nei saggi dipendenti dall'RNA. Qui, presentiamo un metodo pratico, rapido ed economico per estrarre l'RNA da una piccola quantità di tessuto pancreatico non danneggiato.

Estrazione rapida ed economica dell'RNA del tessuto pancreatico ratto

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

Ciao a tutti. Abbiamo progettato, un metodo descrittivo semplice e rapido in classe per un'efficiente estrazione dell'RNA dal tessuto pancreatico. Il principale vantaggio di questo metodo è una procedura ottimizzata per estrarre l'RNA dalla quantità minima di tessuto a causa dell'autolisi. Ciao di nuovo. La dimostrazione della procedura sarà fatta da Sanaz Dastghaib, che è il mio dottorando nel nostro laboratorio presso il Dipartimento di Biochimica dell'Università di Scienze Mediche di Shiraz.Prima prepara il luogo dell'intervento chirurgico, metti tutti i requisiti sotto il cofano. Per inattivare la RNasi sterilizzare tutti gli strumenti chirurgici in forno. Iniettare chetamina-xiloazina intraperitoneale 80/8 milligrammo per chilogrammo. Aggiungere 1 reagente di stabilizzazione cc.RNA in un microtubo adeguato per abitare l'attivazione dell'endonucleasi nei tessuti esistenti. Posizionare immediatamente il topo sulla tavola chirurgica. Allontanati dal chirurgo per un'incisione della linea mediana. Quindi, sterilizzare tutta la superficie addominale con il 70% di alcol per rimuovere i peli del topo. Tagliare la pelle per l'intervento. L'incisione rimodella è stata fatta per aprire l'addome dall'area pubica alle zampe anteriori. Capovolgere gli organi addominali sul lato sinistro, per esporre il pancreas. Trova attentamente il tessuto del pancreas, che si diffonde nella cavità addominale. Individuare l'area esposta per scoprire il pancreas senza essere confusi con il tessuto adiposo. Rimuovere immediatamente il pancreas da 20 a 30 milligrammi dalla cavità addominale in meno di due minuti. Mettere il tessuto rimosso nel microtubo molto rapidamente e tagliarlo con la fresa a cavalco per penetrare nel reagente di stabilizzazione dell'RNA nei tessuti separati. Posizionare il microtubo nel serbatoio dell'azoto per congelare immediatamente. Tutti i loro microtubi devono essere conservati negli 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.

Protocollo ottimizzato per l&#39;estrazione delle proteine ​​dalla valvola mitrale umana

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

Ricerca

Biochimica

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.

Accumulazione del tessuto adiposo del mouse ed elaborazione per l'analisi del RNA

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 analisi di traduzione Iniziazione formazione complessa su mRNAs mammiferi

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.

Rapido isolamento dei Mitoribosome da cellule HEK

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.

Cattura e identificazione delle proteine RNA-leganti utilizzando clic chimica-assistita RNA-Interactoma (Castaldi) strategia di acquisizione

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.

Estrazione ed analisi di propoli verde taiwanesi

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.

Lo studio di RNA interattori della chinasi proteica RNA-attivato durante il ciclo cellulare dei mammiferi

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.

Etichettatura metabolica estremamente rapida e specifica dell'RNA In Vivo con 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.

Estrazione, purificazione e profilatura del ganglioside

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.