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 amounts of input total RNA (e.g. 0.1-1pg/&mu;l), instead of the ng-&mu;g amounts recommended by suppliers.
		<ol>
				<li>We use standard sterile, nuclease-free 200&mu;L thin-walled PCR tubes.</li>
		</ol>
	</li>
	<li>Position RT tubes securely into the micromixer as soon as they are ready for mixing. The micromixer should remain inside the 37&deg;C incubator for the duration of the RT reaction.</li>
	<li>Select the appropriate frequency and amplitude of micromixing. In this example we use 150Hz. </li>
	<li>Switch the micromixer on and leave it on for the entire duration of the RT reaction (60 minutes).</li>
	<li>Switch the micromixer off and take the RT tubes out once the RT reaction is complete. </li>
	<li>Place the RT tubes on ice and proceed as normal with further applications such as quantitative Polymerase Chain Reaction (qPCR or real-time PCR).</li>
</ol>
2. Representative Results: 
The benefit of micromixing in the context of RT reactions is an increase in cDNA yield relative to when other mixing methods (e.g. shaking, vortexing or trituration) are used. This can be seen, for example, upon subsequent analysis of the cDNA using quantitative Polymerase Chain Reaction (qPCR or real-time PCR). Figure 2A illustrates the effects of micromixing for the first 5 minutes (blue, "micromixed5") or the entire 60 minutes (red, "micromixed60") compared with trituration of the RT reaction immediately prior to the RT incubation (black, "trituration"). In this case the volume of the preceding RT reaction was 25&mu;L and it contained 2.5pg of total RNA. qPCR traces using primers designed to detect cDNA representing Nurr-1 mRNA are shown above, and the means &plusmn;SEMs of the number of cycles to reach 50% maximum fluorescence in 3 repeats of this same experiment are plotted below. The asterisk denotes a statistically significant difference (one-way ANOVA with Tukey multiple comparisons). Note that micromixing for the first 5 minutes of the preceding RT reaction produced a non-significant improvement over trituration whereas micromixing for the entire 60 minutes of the preceding RT reaction produced a significant improvement over trituration, the magnitude of which was equivalent to approximately 15 qPCR cycles, on average.
The benefit of micromixing can also be seen in melting curve analysis of the subsequent qPCR products obtained. In the experiment illustrated in Figure 2B, micromixing for the entire 60 minutes of the preceding RT reaction resulted in a consistent qPCR signal in duplicate samples (Figure 2Ba, 2 red traces), whereas micromixing for the first 5 minutes or trituration only produced inconsistent qPCR traces (Figure 2Ba, 2 blue traces and 2 black traces, respectively). The grey traces are negative controls ("neg.") in which cDNA was left out of the qPCR reaction. When the qPCR products from this experiment were denatured by ramping their temperature (i.e. melting curve analysis) it was apparent that appropriate PCR products (i.e. an amplicon that was identical to that obtained when much higher concentrations of mRNA were reverse transcribed and amplified by qPCR, data not shown) were present only following RT reactions that had been micromixed for 60 minutes (Figure 2Bb, 2 red traces). All the others (i.e. micromixed5, trituration and negative controls) generated alternative amplicon products (e.g. primer dimers).
<img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Figure 1"/> Figure 1. Flow diagram of the protocol for applying micromixing to standard laboratory RT reactions. More detailed descriptions of the physical principle involved, the micromixer and how to build an in-house micromixer are provided in references 13,14
<img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Figure 2"/> Figure 2.Representative qPCR data illustrating the benefits of micromixing applied to preceding RT reactions comprising single-cell quantities of mRNA in otherwise standard laboratory RT reactions. A Example qPCR traces detecting cDNA representing mRNA encoding the Nurr-1 gene. The preceding RT reactions contained 2.5pg of total RNA in a volume of 25&mu;L and were performed in an incubator at 37&deg;C for 60 minutes. RT reactions were mixed by trituration of the RT mix prior to incubation (black traces, "trituration"), micromixed during the first 5 minutes of the RT reaction (blue traces, "micromixed5") or micromixed for the entire 60 minutes of the RT reaction (red traces, "micromixed60"). Means &plusmn; SEMs of the number of qPCR cycles required to reach 50% maximum fluorescence are plotted below for n=3 repeats of this experiment. Asterisk indicates a significant difference (one-way ANOVA with Tukey multiple comparisons). B Another qPCR experiment examining Nurr-1 cDNA product, this time following RT reactions containing 25pg of total RNA in a volume of 25&mu;L. qPCR traces (a) as well as melting curve analysis of the qPCR products obtained (b) are shown for the different mixing conditions. Negative controls in which no cDNA was included in the preceding RT reaction are also included (gray traces, "neg."). RT reactions with much higher concentrations of mRNA were also performed and produced melting curve peaks (data not shown) identical to those in (b) for micromixed60 (red traces) samples.

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	<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 authors have nothing to disclose.

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><tbody><tr><td>Total RNA was isolated from snap frozen acutely prepared adult mouse midbrain slices</td><td></td><td></td><td></td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>Arcturus Bioscience</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></td></tr><tr><td>Random hexamer primers</td><td>Promega Corp.</td><td>C1181</td><td></td></tr><tr><td>AMV-RT</td><td>Promega Corp.</td><td>M5101</td><td></td></tr><tr><td>dNTP set</td><td>Promega Corp.</td><td>U1240</td><td></td></tr><tr><td>RNasin&lt;sup&gt;&lt;/sup&gt; Ribonuclease Inhibitor</td><td>Promega Corp.</td><td>N2111</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Promega Corp.</td><td>P1193</td><td></td></tr><tr><td>SYBR&lt;sup&gt;&lt;/sup&gt; Green PCR Master Mix</td><td>Applied Biosystems</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 Spectrophotometer. </td><td>Thermo Fisher Scientific, Inc.</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 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.1K Views.  University of Melbourne. 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.

Video: 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.

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

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.

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 ...

Genetics

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.

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

Neuroscience

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.

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.

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

Biology

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.

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.

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

Developmental Biology

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.

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

Biochemistry

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.

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.

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

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

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Laser Microdissection for Species-Agnostic Single-Tissue Applications