The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTA: E &#39;fondamentale per mantenere condizioni di assenza di RNasi durante l&#39;intera procedura. 1. Adenylation di 3 &#39;Linker <ol><li> Diluire oligonucleotide DNA progettato per le reazioni 3 &#39;legatura a 100 micron di priva di nucleasi H 2 O. NOTA: L&#39;oligonucleotide dovrebbe avere un &#39;gruppo fosfato, un dinucleotide randomizzato al 5&#39; 5 estremità, e un &#39;dideoxycytosine 3. Il 5 &#39;gruppo fosfato è un requisito dell&#39;enzima Mth per un&#39;efficiente 5&#39; adenylation 10. Il fosfato può essere aggiunto un trattamento preliminare del oligonucleotide con un polinucleotide chinasi, o ordinato con la modifica diretta. Il dinucleotide randomizzato all&#39;estremità 5 &#39;è importante per ridurre al minimo la preferenza che ligasi RNA mostre per certo fine-end giunzione reazioni sugli altri 6 sequenza. Il 3 &#39;dideoxycytosine inserisce un elemento di bloccaggio all&#39;estremità 3&#39; estremità del oligonucleotide to inibire la formazione di concatamers linker-linker durante le successive fasi di legatura e serve anche per bloccare &#39;estremità della molecola miRNA-Linker formato alla fine del 3&#39; la reazione di ligazione 11 3. La sequenza di oligonucleotidi linker scelto per il metodo miR-Seq imparziale è la seguente: 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3 &#39;. </li><li> Montare la seguente reazione adenylation: 200 pmol 3 &#39;oligonucleotide linker, 4 microlitri 10x 5&#39; tampone adenylation DNA, 4 microlitri 1 mM ATP, 4 microlitri Mth RNA ligasi, priva di nucleasi H 2 O per un volume finale di 40 microlitri. </li><li> Incubare la reazione a 65 ° C per 1 ora. Terminare la reazione mediante riscaldamento a 85 ° C per 5 min. </li><li> Precipitare linker adenylated con l&#39;aggiunta di 2,5 volumi di etanolo 100% e 1/3 volume di 10 M di acetato di ammonio per rimuovere ATP residuo e per prevenire legature imprevisti nelle reazioni future. <ol><li> In breve, mescolare il algnato di alcool, sale e soluzione oligo all&#39;omogeneità, filatura a piena velocità in una microcentrifuga (~ 15.000 xg) a 4 ° C per 20 min. Lavare il precipitato in etanolo al 70%, aria secca, e risospendere in volume appropriato di priva di nucleasi H 2 O per produrre 50-100 micron di oligo adenylated. </li></ol></li><li> Confermare adenylation eseguendo un gel di poliacrilammide al 18%. <ol><li> Preparare il gel miscelando il seguente insieme: 6,3 g di urea, 6,75 ml di 40% acrilammide, e 1,5 ml di 10x TBE. Aggiungi H 2 O a 15 ml. </li><li> Mescolare fino urea è completamente sciolto e aggiungere 150 microlitri al 10% (w / v) di ammonio persolfato (APS) e 15 microlitri tetramethlyethylenediamine (TEMED). </li><li> Una volta che il gel ha impostato, pre-corsa per 30 min a 24 V / cm di larghezza gel con 1x tampone di corsa TBE a temperatura ambiente. Dopo 30 minuti è da pozzi a filo con tampone di corsa. </li><li> Mescolare 5 pmol di oligonucleotidi adenylated e con uguale volume di tampone di denaturazione 2x RNA di carico (il 95% (v / v) formammide,0,02% (w / v) di SDS, 0,02% blu di bromofenolo (w / v), 0,01% (w / v) Xilene cianolo, 1,0 mM EDTA), portare rapidamente a 75 ° C, e scatto raffreddata su ghiaccio. Dispensare intero campione nel pozzetto del gel. Includere anche un identico ammontare di controllo non adenylated, e gli standard di bassa dimensione peso molecolare. </li><li> Attivare il gel a 24 V / cm fino a quando il blu di bromofenolo è ¾ fino al fondo del gel. Smontare apparecchi, gel post-macchia con soluzione di SYBR-oro 1x a 1x TBE e visualizzare sulla scatola UV-transilluminatore (Figura 2). NOTA: Gel purificare l&#39;oligo adenylated in un modo simile a quello descritto sopra, se &quot;no RNA ingresso&quot; controlli hanno determinato una elevata quantità di prodotto di PCR rispetto ai campioni con ingresso RNA. </li></ol></li></ol> 2. Linker legatura a 3 &#39;Fine di miRNA <ol><li> 3 &#39;legatura <ol><li> Assemblare i seguenti componenti su ghiaccio nell&#39;ordine indicato: 1,0 microlitri 50% (w / v) di PEG 8000, 0,5 &amp;# 956; l 10x tampone RNA ligasi (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTT), 1,0 ml adenylated 3 &#39;Linker (100 mM), inibitore della RNasi 0,5 microlitri, 0,5-8,0 mg di RNA totale, 0,5 microlitri RNA ligasi T4 2, priva di nucleasi H 2 O per un volume finale di 5 microlitri. </li><li> Mescolare la reazione pipettando la soluzione è troppo viscoso a vortice causa dell&#39;elevata concentrazione di PEG 8000. Una volta accuratamente miscelati, posizionare la reazione in un termociclatore con un coperchio riscaldato. Impostare il blocco a 16 ° C, e incubare per 4 ore. NOTA: La reazione può essere scalata fino ad un volume finale di 20 ul senza alcuna perdita di efficienza legatura. Reagire per 4 ore a temperatura ambiente o per una notte a 4 ° C per produrre efficienze legatura sostanzialmente identici. </li></ol></li><li> Gel Purificazione <ol><li> Dopo 3 &#39;legatura, mescolare la reazione con un volume uguale di tampone di caricamento 2x RNA. Riscaldare a 70 ° C per 5 min e scatto raffreddare su ghiaccio. </li&gt; <li> Eseguire il campione su un gel di poliacrilammide al 15% per la purificazione PAGE. <ol><li> Preparare un gel di poliacrilammide al 15% contenente 7 M urea, in modo simile a quello descritto in precedenza al punto 1.5. </li><li> Pre-eseguire il gel a 24 V / cm di larghezza gel per almeno 30 min. Spegnere l&#39;alimentazione elettrica, pozzi a filo, campione del carico e gel di corsa a stessa tensione pre-run. </li><li> Caricare il campione e eseguire la pagina fino a quando il blu di bromofenolo è appena uscito il gel. Il prodotto desiderato migrerà vicino al cyanol xilene. </li></ol></li></ol></li><li> Colorazione, visualizzazione, e l&#39;escissione <ol><li> Smontare apparecchi gel e posto gel in vassoio pulito con 1x tampone di corsa (TBE), 1x SYBR-oro, e di roccia per 15 min. </li><li> Rimuovere il gel dal piatto macchie e visualizzare su scatola transilluminatore UV coperta con pellicola trasparente pulito. Il prodotto legatura dovrebbe essere di circa 45 nucleotidi di lunghezza. </li><li> Accise la regione contenente il prodotto con una legatura Razo pulitolama r e il luogo in 0,5 ml di tubo di micro-centrifuga. </li></ol></li><li> Isolamento del prodotto e Precipitazioni <ol><li> Pierce fondo della provetta da 0,5 ml microcentrifuga con un ago da 30 G e posizionare il tubo forato in un secondo pulita, a bassa ritenzione, 1,5 ml provetta. Centrifugare per 5 minuti a ~ 15.000 x g. </li><li> Visivamente che l&#39;intera fetta del gel è spostato attraverso il foro nel tubo interno. Se necessario, usare un puntale pulito per spingere alcuni frammenti di gel più vicino alla buca. </li><li> Smaltire il tubo vuoto 0,5 ml e aggiungere 400 ml di alto contenuto di sale tampone colonna (HSCB 400 mM NaCl, 25 mM Tris-HCl pH 7.5, 0.1% SDS) per il liquame acrilamide. Tubo di roccia (s) a 4 ° C per una notte. </li><li> Trasferimento liquami di una colonna di spin acetato di 0,22 micron di cellulosa con un puntale ampio tunnel. Centrifugare per 3 minuti a temperatura ambiente a ~ 15.000 xg per raccogliere tutto il liquido dalla matrice di gel di acrilammide. </li><li> Trasferire il liquido di un ambiente pulito, bassa ritenzione, provetta da 1,5 ml contieneing 1 ml di 20 mg / ml di glicogeno. Aggiungere un uguale volume di isopropanolo e 1/10 di volume di acetato di sodio. Miscelare soluzione capovolgendo tubo più volte il precipitato in -80 ° C freezer per 20 min. </li><li> Centrifugare a piena velocità a 4 ° C per 20 minuti per far sedimentare l&#39;acido nucleico. Rimuovere il surnatante e lavare in etanolo al 70%. Aria pellet secco per 10 minuti a temperatura ambiente e procedere direttamente al 5 &#39;passo legatura. </li></ol></li></ol> 3. Linker legatura a 5 &#39;Fine del miRNA-3&#39; Linker Hybrid <ol><li> Per l&#39;acido nucleico in granuli, aggiungere i seguenti componenti: 2 microlitri PEG 8000 (50% w / v), 0,5 ml 10x RNA Ligase Buffer, 0,5 microlitri ATP (10 mM), 0,5 microlitri NN-RNA oligo (100 mM), e H 2 O ad un volume finale di 4 microlitri. NOTA: La composizione del &#39;NN-RNA oligo&#39; è il seguente 5&#39;-invertita dideossi-TCUACAGUCCGACGAUCNN3 &#39;ed è stato purificato tramite HPLC dal costruttore. </li><li> Mescolarequesto campione pipettando su e giù più volte. Fare questo per un certo tempo (fino a 5 minuti) per garantire che il pellet è stato completamente ri-solubilizzati. Se ri-solubilizzazione è problematico, riscaldare brevemente campione (3-5 min) a 37 ° C e riprendere pipettaggio mescolare. </li><li> Una volta che il pellet è tornato in soluzione, trasferire il contenuto in una provetta pulita PCR contenente 1 ml di una miscela 1: 1 di inibitore della RNasi e T4 RNA ligasi 1 (5 unità). Anche in questo caso mescolare pipettando su e giù. </li><li> Incubare la reazione in un termociclatore a 37 ° C per 4 ore. Procedere immediatamente alla sintesi del DNA. </li></ol> 4. trascrizione inversa / cDNA Synthesis <ol><li> Aggiungere i seguenti componenti al 5 &#39;reazione Legatura: 2 microlitri di buffer 5x RT, 0,75 ml 100 mM DTT, 1 ml Illumina RT Primer 5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39; (1 micron), e 0,5 microlitri dNTP (10 mm). </li><li> Mescolare pipettando e calore a 65 ° C per 5 min. Poi raffreddare lentamente in panchina top. </li><li> Una volta che la reazione è raffreddata a temperatura ambiente, il tubo posto su ghiaccio e aggiungere 0,5 ml ciascuno di enzima trascrittasi inversa e inibitori RNasi. Mescolare pipettando e posto in termociclatore per la fase di estensione indicato nelle istruzioni del produttore. </li><li> Una volta completato Aggiungere 5 ml di priva di nucleasi H 2 O per portare il volume finale della libreria di cDNA fino a 15 ml. </li></ol> 5. Biblioteca Preparazione <ol><li> Per l&#39;indicizzazione PCR, montare la seguente reazione su ghiaccio: 6 ml 5x Phusion Buffer, 1 microlitri dNTP (10 mm), 0,9 ml di DMSO, 0,75 ml 5 &#39;Primer (25 micron), 0,75 ml 3&#39; Primer (25 micron), biblioteca 3 microlitri di cDNA (concentrazione variabile a seconda della quantità di input), 0,5 ml Phusion Polymerase (1 unità), priva di nucleasi H 2 O per un volume finale di 30 ml. Scegli le sequenze dei primer per garantire la compatibilità con il modello e la piattaforma di sequenziamentoscelta. <ol><li> Eseguire la reazione di PCR con le seguenti impostazioni: denaturazione iniziale a 98 ° C per 3 minuti, per i primi 4 cicli, denaturare 98 ° C per 15 sec, ricottura a 50 ° C per 15 sec, estendere a 72 ° C per 15 sec . NOTA: A seconda della quantità del materiale in ingresso, variare il totale numero di cicli di PCR. Tipicamente, per un ingresso di 1-2 mg di RNA totale, 13 cicli di PCR in totale sono sufficienti a generare la libreria miR-Seq. </li><li> Effettuare cicli successivi, ad esempio, cicli 5-12, in un modo a due fasi in cui i cicli di ricottura e di estensione sono combinati e effettuate a 70 ° C per 30 sec e la denaturazione è ancora 98 ° C per 15 sec. NOTA: Il volume di reazione è di 30 ml per consentire una pausa il programma PCR e rimuovere una aliquota di 10 ml al numero di cicli predeterminati per monitorare l&#39;aspetto del prodotto desiderato. Tipicamente, i cicli di 10, 13, e 16 sono scelti per il test di un campione di RNA totale. </li></ol></li><li> Preparareun 8% non denaturante acrilamide gel miscelando 2 ml di 40% acrilammide, 1 ml di 10x TBE, e H 2 O a 10 ml. Quindi aggiungere 100 ml di 10% di APS e 10 ml di TEMED. Versare il gel e lasciare set. <ol><li> Pre-eseguire il gel a 12 V / cm di larghezza gel per 30 min. Pozzi a filo. Aggiungere 1 ml di 10x tampone di caricamento (15% Ficoll-400, 66 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% (w / v) di SDS, 0,01% (w / v) di blu di bromofenolo), aggiungere 10 ml di campione a ciascun pozzetto di dimensioni 5 millimetri x 1 mm x 15 mm. In genere, ogni bene può caricare fino a 30 ml di campioni. Eseguire il gel campione a 10 V / cm di larghezza gel 1x tampone TBE finché il colorante blu di bromofenolo appena esaurisce il fondo del gel. </li><li> Colorare e visualizzare il gel come descritto al punto 2.3. </li><li> Accise la coppia di banda di 146 base e eluire durante la notte in 0,25 ml di HSCB in modo simile a quanto descritto nella sezione 2.4. Precipitare il DNA in un uguale volume di isopropanolo e 1/10 di volume di acetato di sodio. Lavare pellet nel 70% eTHANOL e risospendere in tampone TE. </li><li> Quantificare la libreria utilizzando PicoGreen secondo le raccomandazioni del produttore. </li></ol></li></ol>

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The authors declare no competing financial interests.

La metodologia qui descritta si avvale di diverse variabili chiave per massimizzare l&#39;efficienza di legatura, ovvero alte concentrazioni di PEG, l&#39;uso di linker randomizzati, e alta concentrazione di linker 2,6,9. Questo approccio permette di librerie di sequenziamento in modo affidabile quantitativi di campioni di RNA totale di 2. Abbiamo condotto molteplici titolazioni di ingresso RNA e hanno concluso che il metodo precedente è più adatto per importi totali di RNA nel range 1-8 mg (dati...

Metodologie basate High-throughput sequencing sono stati ampiamente applicati a molti campioni biologici negli ultimi anni notevolmente ampliando la nostra comprensione della complessità molecolare dei sistemi biologici 3,4. Tuttavia, la preparazione di campioni di RNA per high-throughput sequencing conferisce spesso pregiudizi specifici inerenti alla metodologia utilizzata, che limitano la potenziale utilità di queste tecniche potenti. Questi pregiudizi metodo specifico sono stati ben documentati per la base di legatura-piccolo-RNA preparati biblioteca 1,2,5,6. Questi pregiudizi in una modifica di 1000 volte in legge dei numeri per miRNA sintetici equimolari, facendo inferenza di miRNA abbondanza di dati di sequenziamento selvaggiamente variabile e soggetto a errori. Gli studi si concentrano sulle proprietà di T4 ligasi RNA fago-derivati ​​hanno documentato che gli enzimi mostrano preferenze di base nucleotide-7, che si manifestano come le biblioteche di parte negli esperimenti di sequenziamento high-throughput <sup&gt; 1,2,8. Per minimizzare le distorsioni impartite dalla RNA ligasi, molteplici strategie sono state impiegate; macromolecolare affollamento 9, randomizzare la sequenza nucleotidica sull&#39;adattatore che è prossimale al sito legatura 6, e utilizzando alte concentrazioni di adattatore legatura 2. Attraverso una combinazione di questi tre approcci abbiamo sviluppato un flusso di lavoro per la preparazione imparziale di piccole librerie di RNA compatibili per high-throughput sequencing (Figura 1). Per i confronti diretti tra i protocolli correnti e il nostro metodo ottimizzato, si prega di fare riferimento al nostro recente rapporto 2. Questo metodo ottimizzato produce efficienze di legatura superiore al 95% sia a 3 &#39;e 5&#39; passi e permette la legatura imparziale di piccole molecole di RNA da campioni sintetici e biologici 2.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

Mirna clonazione e high-throughput sequencing, chiamati miR-Seq, si trova solo come un approccio a livello di trascrittoma per quantificare miRNA con risoluzione di singolo nucleotide. Questa tecnica cattura miRNA collegando adattatori 3 &#39;e 5&#39; oligonucleotide alle molecole miRNA e permette la scoperta de novo miRNA. Accoppiamento con potenti piattaforme di sequenziamento di nuova generazione, miR-Seq è stato determinante per lo studio della biologia miRNA. Tuttavia, distorsioni significative introdotte dal oligonucleotide passi legatura hanno impedito miR-Seq da essere impiegato come strumento di quantificazione accurata. Studi precedenti hanno dimostrato che i pregiudizi in attuali metodi di miR-Seq spesso portano a inesatte miRNA quantificazione di errori fino a 1.000 volte per alcuni miRNA 1,2. Per risolvere questi pregiudizi impartite dalla RNA legatura, abbiamo sviluppato un piccolo metodo di legatura RNA che si traduce in efficienza legatura di oltre il 95% sia per la 3 &#39;e 5&#39; passi legatura. Benchmarking questo imdimostrato metodo di costruzione libreria utilizzando miRNA sintetici equimolare o differenziale misti, produce sempre legge i numeri con deviazione inferiore a due volte rispetto al valore atteso. Inoltre, questo metodo ad alta efficienza miR-Seq permette accurate genome-wide miRNA profiling dall importo totale di campioni in vivo di RNA 2.

I risultati attesi per il metodo precedente dovrebbe inizialmente essere l&#39;osservazione di un solo turno nucleotide (aumento) delle dimensioni del oligonucleotide DNA che è stato oggetto di adenylation da Mth RNA ligasi (Figura 2). Dopo 3 &#39;legatura, visualizzazione del gel di acrilammide indica (vedi Figura 3) taglienti alte bande peso molecolare evidenti nella regione di 100-300 nucleotidi del gel. Ciò indica che il campione di RNA totale utilizzato è di alta qualit...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Legatura altamente efficiente delle piccole molecole di RNA per microRNA Quantificazione mediante sequenziamento High-Throughput

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K Views.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

Video: Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidina a base di fluorescenza di analisi, per determinare piccole molecole Potenziale Modifica delle proprietà del doppio strato lipidico

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Ricerca

Biologia

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening per la piccola molecola modulatori di canali di potassio Inward Rectifier

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Generazione Substrato per endonucleasi di CRISPR / Cas Sistemi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manuale Schermo piccole molecole si avvicina ad alta velocità Uso embrioni di zebrafish

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Automatizzata Modular High Throughput Screening esopolisaccaride piattaforma Accoppiato con altamente sensibile Analisi Carboidrati impronte digitali

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: Genome-wide Mappatura delle interazioni proteina-DNA da High Throughput Sequencing di Adenina-metilato frammenti di DNA

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Un protocollo per la valutazione funzionale del Whole-Protein Saturazione mutagenesi biblioteche Utilizzando High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...