Diese Studie wurde von der National Health and Medical Research Council of Australia (Projekt Grant No. 6288480) und der Scobie und Clare MacKinnon Trust unterstützt.

1. Mikrovermischung RT Reaction <ol><li> Vor der Durchführung einer RT-Reaktion mit Mikrovermischung, bringen die Mikromischer auf die gewünschte Temperatur der RT-Reaktion. <ol><li> Legen Sie die Mikromischer in einem 37 ° C (oder die Temperatur durch die RT Lieferant empfohlen) Inkubator für mindestens 1 Stunde vor Beginn der RT-Reaktion. </li></ol></li><li> Richten Sie die RT-Mix nach dem Reverse-Transkriptase-Lieferanten (zB MMTV-RT von Promega, Omniscript von Qiagen) Anweisungen, außer Nutzung Single-Cell-Mengen-Eingang Gesamt-RNA (z. B. 0.1-1pg/μl), anstelle der ng-pg Beträge, die Lieferanten zu empfehlen. <ol><li> Wir verwenden Standard-sterile, Nuklease-freies 200 ul dünnwandigen PCR-Gefäße. </li></ol></li><li> Position RT Rohre sicher in den Mikromischer, sobald sie bereit sind für das Mischen sind. Die Mikromischer sollte innerhalb der 37 ° C Inkubator für die Dauer der RT-Reaktion bleiben. </li><li> Wählen Sie die gewünschte Frequenz und Amplitude Mikrovermischung. In diesem Beispiel verwenden wir 150Hz. </li><li> Schalten Sie den Mikromischer auf und lassen Sie es für die gesamte Dauer der RT-Reaktion (60 Minuten). </li><li> Schalten Sie den Mikromischer aus und nehmen Sie die RT Rohre aus, wenn die RT-Reaktion abgeschlossen ist. </li><li> Legen Sie die RT Röhrchen auf Eis, und fahren wie gewohnt mit weiteren Anwendungen wie quantitative Polymerase-Kettenreaktion (qPCR oder real-time PCR). </li></ol> 2. Repräsentative Ergebnisse: Der Vorteil der Mikrovermischung im Rahmen der RT-Reaktionen ist eine Erhöhung der cDNA Ausbeute bezogen auf, wenn andere Mischverfahren (zB Zittern, Vortexen oder Verreibung) verwendet werden. Dies zeigt sich zum Beispiel bei der anschließenden Analyse der cDNA mittels quantitativer Polymerase-Kettenreaktion (qPCR oder real-time PCR) werden. Abbildung 2A zeigt die Auswirkungen der Mikrovermischung für die ersten 5 Minuten (blau, &quot;micromixed5&quot;) oder den gesamten 60 Minuten (rot, &quot;micromixed60&quot;), verglichen mit Verreibung der RT-Reaktion unmittelbar vor der RT Inkubation (schwarz, &quot;Verreiben&quot; ). In diesem Fall wird die Lautstärke des vorangegangenen RT-Reaktion wurde 25 &amp;mgr; l, und es enthielt 2.5pg von Gesamt-RNA. qPCR Spuren unter Verwendung der Primer entwickelt, um cDNA repräsentiert Nurr-1 mRNA erkennen sind oben gezeigt, und die Mittelwerte ± SEM der Anzahl der Zyklen bis 50% maximale Fluoreszenz in 3 Wiederholungen desselben Experiments zu erreichen sind nachfolgend dargestellt. Das Sternchen bedeutet einen statistisch signifikanten Unterschied (one-way ANOVA mit Tukey multiple Vergleiche). Beachten Sie, dass Mikrovermischung für die ersten 5 Minuten des vorangegangenen RT-Reaktion eine nicht-signifikante Verbesserung gegenüber Verreibung hergestellt, während Mikrovermischung für den gesamten 60 Minuten der vorhergehenden RT-Reaktion zu einer signifikanten Verbesserung gegenüber Verreibung, deren Größe entsprach etwa 15 qPCR Zyklen im Durchschnitt. Der Vorteil der Mikrovermischung kann auch in Schmelzkurvenanalyse der nachfolgenden qPCR erhaltenen Produkte gesehen werden. In dem Experiment in Abbildung 2B dargestellt, Mikrovermischung für den gesamten 60 Minuten der vorhergehenden RT-Reaktion führte zu einer konsequenten qPCR-Signal in zwei Proben (Abbildung 2Ba, 2 rote Spuren), während Mikrovermischung für die ersten 5 Minuten oder Verreibung nur produziert inkonsistent qPCR Spuren (Abbildung 2Ba, 2 blaue Spuren und 2 schwarzen Spuren, jeweils). Der graue Spuren sind negative Kontrollen (&quot;neg.&quot;), In der cDNA aus der qPCR-Reaktion wurde nach links. Wenn die qPCR Produkten aus diesem Experiment wurden denaturiert durch Rampen ihre Temperatur (dh Schmelzkurvenanalyse) war es offensichtlich, dass geeignete PCR-Produkte (dh ein Amplikon derjenigen identisch ist, erhält man, wenn viel höhere Konzentrationen des mRNA revers transkribiert wurden und durch qPCR amplifiziert wurde, Daten nicht gezeigt) waren nur folgende RT Reaktionen, die für 60 Minuten (Abbildung 2Bb, 2 rote Spuren) war micromixed. Alle anderen (dh micromixed5, Zerreiben und negative Kontrollen) generiert alternative Amplikon Produkte (zB Primer-Dimere). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Fließschema des Protokolls für die Anwendung Mikrovermischung Standard-Labor-RT-Reaktionen. Genauere Beschreibungen des physikalischen Prinzips beteiligt, die Mikromischer und wie eine in-house Mikromischer bauen in Verweise 13,14 zur Verfügung gestellt <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Representative qPCR-Daten, welche die Vorteile der Mikrovermischung zu vorangegangenen RT-Reaktionen mit Single-Cell-Mengen von mRNA in sonst üblichen Labor-RT-Reaktionen eingesetzt. Ein Beispiel qPCR Spuren erkennen cDNA repräsentiert mRNA codiert Nurr-1-Gen. Die vorstehenden RT Reaktionen enthielten 2.5pg von Gesamt-RNA in einem Volumen von 25 &amp;mgr; l und wurden in einem Brutschrank bei 37 ° C für 60 Minuten durchgeführt. RT-Reaktionen wurden durch Verreiben der R gemischtT-Mix vor der Inkubation (schwarz Spuren &quot;, Verreibung&quot;), während der ersten 5 Minuten der RT-Reaktion (blau Spuren &quot;, micromixed5&quot;) micromixed oder micromixed für den gesamten 60 Minuten der RT-Reaktion (rot Spuren &quot;, micromixed60&quot; ). Mittelwert ± SEM der Anzahl der qPCR-Zyklen erforderlich, um 50% maximale Fluoreszenz zu erreichen sind nachfolgend für n = 3 dargestellt Wiederholungen dieses Experiments. Asterisk zeigt einen signifikanten Unterschied (one-way ANOVA mit Tukey multiple Vergleiche). B Ein weiterer qPCR Experiment untersucht Nurr-1 cDNA-Produkt, diesmal nach RT-Reaktionen, die 25pg von Gesamt-RNA in einem Volumen von 25 &amp;mgr; l. qPCR Spuren (a) sowie Schmelzkurvenanalyse der qPCR Produkten erhalten (b) sind für die verschiedenen Misch-Bedingungen gezeigt. Negative Kontrollen, in denen keine cDNA in den vorangegangenen RT-Reaktion enthalten war sind ebenfalls enthalten (graue Spuren &quot;, neg.&quot;). RT-Reaktionen mit sehr viel höheren Konzentrationen von mRNA wurden ebenfalls durchgeführt und produziert Schmelzkurve Peaks (Daten nicht gezeigt) identisch mit denen in (b) für micromixed60 (rote Kurven) Proben.

<ol>
	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., Quake, 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=.">.</a> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., Quake, 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=.">.</a> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

Die Autoren haben nichts zu offenbaren.

Die Methode der Anwendung von Mikrovermischung Standard-Labor-RT hier beschriebenen Reaktionen kann natürlich beinhalten mRNA geerntet über einen beliebigen Methode (zB Zell-Lyse-, Laser-Mikrodissektion). Es kann auch eine beliebige Marken oder Typen von RT-Reagenzien, bei jeder Temperatur (innerhalb der Toleranz von den Materialien der Mikromischer) und einen beliebigen Zeitraum. Zum Beispiel haben wir eine verbesserte cDNA Erträge aus RT-Reaktionen mit random Hexamer oder Oligo-dT-Primer beobachtet. Es könnte auch...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Gesamt-RNA wurde aus schockgefroren akut vorbereitet erwachsenen Maus Mittelhirn Scheiben isoliert </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA Isolation Kit </td><td> Arcturus, CA, USA </td><td> KIT0204 </td><td> Das Kit ist ab sofort bei Applied Biosystems </td></tr><tr><td> DNA-freie DNase Behandlung und Beseitigung Reagenzien </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Zufällige Hexamer Primer </td><td> Promega </td><td> C1181 </td><td></td></tr><tr><td> AMV-RT </td><td> Promega </td><td> M5101 </td><td></td></tr><tr><td> dNTP Set </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasin Ribonuclease Inhibitor </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Nuklease-freies Wasser </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Green PCR Master Mix </td><td> Applied Biosystem </td><td> 4309155 </td><td></td></tr><tr><td> HPRT forward (20mer): CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> HPRT reverse (20mer): TAT GTC CCC CGT TGA CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 forward (23mer): CAG CTC CGA TTT CTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 reverse (23mer): GGT GAG GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 Spektralphotometer. </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Corbett Rotor Gene RG-6000 </td><td> Corbett Life Science </td><td></td><td> Corbett Life Science wurde von QIAGEN im Juli 2008 erworben </td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

Die Korrelation der Genexpression mit Zellverhalten ist ideal an den Single-Cell-Ebene getan. Dies ist jedoch nicht leicht zu erreichen, weil die geringe Menge an labile mRNA in einer einzigen Zelle (1-5% der 1-50pg Gesamt-RNA oder 0,01-2.5pg mRNA, pro Zelle 1) meist abbaut, bevor sie transkribiert werden rückgängig machen kann in eine stabile cDNA-Kopie. Zum Beispiel unter Verwendung von Standard-Labor Reagenzien und Hardware können nur eine kleine Anzahl von Genen qualitativ pro Zelle 2 bewertet. Eine Möglichkeit, die Effizienz der Standard-Labor-Reverse-Transkriptase (RT)-Reaktionen (zB Standard-Reagenzien in mikrolitergroßen) mit Single-Cell-Mengen von mRNA zu erhöhen wäre, schneller zu mischen Reagenzien, so dass die mRNA in cDNA umgewandelt werden können, bevor es abgebaut wird. Dies ist jedoch nicht trivial, weil bei Mikroliter Flüssigkeit Skalen Strömung laminar, dh derzeit verfügbaren Methoden der Durchmischung (dh Zittern, Vortexen und Verreibung) nicht genügend chaotische Bewegung effektiv Reagenzien zu produzieren. Um dieses Problem zu lösen, haben Mikro-Maßstab Mischtechniken verwendet 3,4 sein. Eine Reihe von mikrofluidischen-basierte Misch-Technologien entwickelt worden, die erfolgreich erhöhen RT Reaktionsausbeuten 5-8. Allerdings erfordern Mikrofluidik-Technologien spezialisierte Hardware, die relativ teuer und noch nicht allgemein verfügbar ist. Eine billigere, bequemere Lösung ist wünschenswert. Das Hauptziel dieser Studie ist es zu demonstrieren, wie Einsatz eines neuartigen &quot;Mikrovermischung&quot; Technik, um Standard-Labor-RT-Reaktionen mit Single-Cell-Mengen von mRNA signifikant erhöht ihre cDNA ergibt. Wir finden cDNA Erträge um etwa 10 bis 100-fache, die ermöglicht zu erhöhen: (1) eine größere Anzahl von Genen pro Zelle analysiert werden, (2) mehr quantitative Analyse der Genexpression, und (3) eine bessere Erkennung von Low-Fülle Gene in einzelnen Zellen. Die Mikrovermischung auf akustische Microstreaming 9-12, ein Phänomen, wo die Schallwellen ausbreiten um ein kleines Hindernis zu schaffen eine mittlere Strömung in der Nähe des Hindernisses auf. Wir haben eine akustische Microstreaming-basierten Gerät (&quot;Mikromischer&quot;) mit einer Schlüssellänge Vereinfachung entwickelt, akustische Microstreaming kann bei Audio-Frequenzen, indem sichergestellt wird das System über einen Flüssigkeits-Luft-Schnittstelle mit einem kleinen Krümmungsradius 13 erreicht werden. Der Meniskus einer Mikroliter Volumen der Lösung in einer Röhre bietet eine entsprechend kleinen Krümmungsradius. Die Verwendung von Audio-Frequenzen bedeutet, dass die Hardware kann kostengünstig und vielseitig 13 und Nukleinsäuren und anderen biochemischen Reagenzien dürfen nicht beschädigt sein, wie sie mit Standard-Labor sonicators werden kann.

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Wir beschreiben ein neuartiges Verfahren zur Erhöhung der cDNA-Ausbeute von Single-Cell-Mengen von mRNA in sonst üblichen Labor reverse Transkription Reaktionen. Die Neuheit besteht in der Verwendung von einem Mikromischer, die das Phänomen der akustischen Microstreaming nutzt, um Flüssigkeiten bei Mikroliter Skalen effektiver Mix als Schütteln, Vortexen oder Verreibung.

Steigende cDNA Erträge aus Einzel-Zell-Mengen von mRNA in Standard Laboratory Reverse-Transkriptase-Reaktionen mit Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.1K Aufrufe.  University of Melbourne. Wir beschreiben ein neuartiges Verfahren zur Erhöhung der cDNA-Ausbeute von Single-Cell-Mengen von mRNA in sonst üblichen Labor reverse Transkription Reaktionen. Die Neuheit besteht in der Verwendung von einem Mikromischer, die das Phänomen der akustischen Microstreaming nutzt, um Flüssigkeiten bei Mikroliter Skalen effektiver Mix als Schütteln, Vortexen oder Verreibung.

Serie: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

High Throughput MicroRNA Profiling: Optimierte Multiplex qRT-PCR in Nanoliter-Maßstab auf dem Fluidigm Dynamische ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Forschung

Biotechnik

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Anpassung der 3' endet rasche Verstärkung der CDNA Transkripte in Krebs zuordnen

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

Genetik

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Transkriptom-Analyse von Einzelzellen

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neurowissenschaften

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 &#176;C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5&#8217;-centric expression profiling.

Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5&#8217;ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

Transkription Start Site Mapping mit Super-low Input Carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Analyse von Genexpressionsdaten aus marinen mikrobiellen Gemeinschaften mit Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Biologie

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

Beziehen High Quality-RNA aus Einzel-Zellpopulationen in menschlichen Postmortem Gehirngewebe

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Multiplexed Single Cell mRNA Sequenzierungsanalyse von Mausembryonalzellen

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Entwicklungsbiologie

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

Untersuchung der RNA-Struktur mit Dimethylsulfat-Mutationsprofilierung mit Sequenzierung in vitro und in Zellen

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Biochemie

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

Einzelne Zelle Multiplex reversen Transkription Polymerase-Kettenreaktion nach Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Laser-Mikrodissektion für speziesunabhängige Einzelgewebeanwendungen

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...