This study was supported by the National Health and Medical Research Council of Australia (project grant no. 6288480) and the Scobie and Clare MacKinnon Trust.

1. Micromixing an RT Reaction
<ol>
	<li>Before performing an RT reaction with micromixing, equilibrate the micromixer to the desired temperature of the RT reaction.
		<ol>
				<li>Place the micromixer inside a 37&deg;C (or the temperature recommended by 	the RT supplier) incubator for at least 1 hour prior to the onset of the RT 	reaction.</li>
		</ol>
	</li>
	<li>Set up the RT mix according to the reverse transcriptase supplier's (e.g. MMTV-RT from Promega, Omniscript from Qiagen) instructions, except use single-cell ...

<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>

The method of application of micromixing to standard laboratory RT reactions described here can, of course, involve mRNA harvested via any method (e.g. cell lysis, laser capture microdissection). It can also comprise any brands or types of RT reagents, any temperature (within the tolerance of the materials of the micromixer), and any period of time. For example, we have observed improved cDNA yields from RT reactions comprising random hexamer or oligo-dT primers. It might also be applied to other biochemical transform...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent	</th>
			<th>Company	</th>
			<th>Catalogue number	</th>
			<th>Comments (optional)</th>
		</tr>
		
		<tr>
			<td>Total RNA was isolated from snap frozen acutely prepared adult mouse midbrain slices</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>PicoPure RNA Isolation Kit	</td>
			<td>Arcturus, CA, USA	</td>
			<td>KIT0204	</td>
			<td>The kit is now available from Applied Biosystems</td>
		</tr>
		
		<tr>
			<td>DNA-free DNase Treatment and Removal Reagents	</td>
			<td>Ambion	</td>
			<td>AM1906M</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Random hexamer primers	</td>
			<td>Promega	</td>
			<td>C1181</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>AMV-RT	</td>
			<td>Promega	</td>
			<td>M5101</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>dNTP set	</td>
			<td>Promega	</td>
			<td>U1240</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>RNasin Ribonuclease Inhibitor	</td>
			<td>Promega	</td>
			<td>N2111</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Nuclease-Free Water	</td>
			<td>Promega	</td>
			<td>P1193</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>SYBR Green PCR Master Mix	</td>
			<td>Applied Biosystem	</td>
			<td>4309155</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Hprt forward (20mer):CTT TGC TGA CCT GCT GGA TT</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Hprt reverse (20mer):TAT GTC CCC CGT TGA CTG AT</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Nurr1 forward (23mer):CAG CTC CGA TTT CTT AAC TCC AG</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Nurr1 reverse (23mer):GGT GAG GTC CAT GCT AAA CTT GA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>NanoDrop 1000 Spectrophotometer. </td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Corbett Rotor Gene RG-6000</td>
			<td>Corbett Life Science</td>
			<td>&nbsp;</td>
			<td>Corbett Life Science was acquired by QIAGEN in July 2008</td>
		</tr>
		
	</tbody>
</table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using 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 labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

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

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

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

Research

JoVE Journal

Bioengineering

10.1K vues.  University of Melbourne. Nous décrivons une nouvelle méthode pour augmenter le rendement d'ADNc de cellule unique quantités d'ARNm dans les réactions de laboratoire standard de transcription inverse contraire. La nouveauté réside dans l'utilisation d'un micromélangeur, qui utilise le phénomène de la microstreaming acoustique, de mélanger les liquides à l'échelle du microlitre plus efficacement que secouer, vortex ou trituration.