Name: Transcriptome Analysis of Single Cells (Video) | JoVE Journal
Uploaded: 2011-04-25T14:30:00.000+00:00
Duration: 7 min 27 s
Description: Learn single-cell transcriptome analysis using aRNA amplification for neuroscience research with rat primary neuronal cultures. This method enables detailed study of gene expression variability between individual neurons.

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5 minutes

Thank you to Kevin Miyashiro for plating and maintaining cell cultures, to Dr. Terri Schochet for providing cell cultures for the pictures included in this document. In addition, thank you to Kevin Miyashiro, Dr. Peter Buckley, and Tiina Pertiz for input on the aRNA procedure. Funding for this work was from the National Institute on Aging, the National Institute on Mental Health and the Human Resources Fact Finder funds from the Commonwealth of Pennsylvania.

<ol><li>Davis, J. E., Eberwine, J. H., Hinkle, D. A., Marciano, P. G., Meaney, D. F., McIntonsh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodological+considerations+regarding+single-cell+gene+expression+profiling+for+brain+injury%5BTitle%5D)+AND+%22Neurochemical+Research%22%5BJournal%5D)">Methodological considerations regarding single-cell gene expression profiling for brain injury.</a> Neurochemical Research. 29, 1113-1121 (2004).</li>
<li>Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+molecular+biology%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Single-cell molecular biology.</a> Nature Neuroscience. 4, 1155-1156 (2001).</li>
<li>Kamme, F., Salunga, R., Yu, J., Tran, D., Zhu, J., Luo, J., Bittner, A., Guo, H., Miller, N., Wan, J., Erlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+microarray+analysis+in+hippocampus+CA1%3A+Demonstration+and+Validation+of+Cellular+Heterogeneity%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Single-cell microarray analysis in hippocampus CA1: Demonstration and Validation of Cellular Heterogeneity.</a> The Journal of Neuroscience. 23, 3607-3607 (2003).</li>
<li>Hinkle, D., Glanzer, J., Sarabi, A., Pajunen, T., Zielinski, J., Belt, B., Miyashiro, K., McIntosh, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+neurons+as+experimental+systems+in+molecular+biology%5BTitle%5D)+AND+%22Progress+in+Neurobiology%22%5BJournal%5D)">Single neurons as experimental systems in molecular biology.</a> Progress in Neurobiology. 72, 129-142 (2004).</li>
<li>Ginsberg, S. D., Elarova, I., Ruben, M., Tan, F., Counts, S. E., Eberwine, J. H., Trojanowski, J. Q., Hemby, S. E., Mufson, E. J., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+gene+expression+analysis%3A+Implications+for+Neurodegenerative+and+Neuropsychiatric+Disorders%5BTitle%5D)+AND+%22Neeurochemical+Research%22%5BJournal%5D)">Single-cell gene expression analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders.</a> Neeurochemical Research. 29, 1053-1064 (2004).</li>
<li>Eberwine, J., Spencer, K., Miyashirto, K., Mackler, S., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complementary+DNA+synthesis+in+situ%3A+methods+and+applications%5BTitle%5D)+AND+%22Methods+Enxymol%22%5BJournal%5D)">Complementary DNA synthesis in situ: methods and applications.</a> Methods Enxymol. 216, 80-100 (1992).</li>
<li>Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., Coleman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+gene+expression+in+single+live+neurons%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Analysis of gene expression in single live neurons.</a> Proc. Natl. Acad. Sci. USA. 89, 3010-3014 (1992).</li>
<li>Kelz, M. B., Dent, G. W., Therianos, S., Marciano, P. G., McIntosh, T. K., Coleman, P. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+antisense+RNA+amplification+and+microarray+analysis+as+a+tool+for+studying+neurological+degeneration+and+restoration%5BTitle%5D)+AND+%22Sci.+Aging+Knowledge+Eviron%22%5BJournal%5D)">Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.</a> Sci. Aging Knowledge Eviron. 1, re1-re1 (2002).</li>
<li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+hippocampal+neurons+in+dispersed+cell+culture%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126, 397-425 (1977).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22P-1000+%26+P-97+Pipette+Cookbook%22">P-1000 & P-97 Pipette Cookbook</a>. , rev. F, Sutter Instrument. (2010).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22MinElute+Handbook+For+MinElute+Reaction+Cleanup+Kit%22">MinElute Handbook For MinElute Reaction Cleanup Kit</a>. , Qiagen. 28-29 (2004).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Megascript+kit%22">Megascript kit</a>. , Ambion. 7-8 Forthcoming.</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22MEGAclear+kit%22">MEGAclear kit</a>. , Applied Biosystems. 4-5 Forthcoming.</li>
<li>Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mRNA+expression+analysis+of+tissue+sections+and+single+cells%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li>
<li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li>
<li>Miyashiro, K., Dichter, M., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis%3A+Implications+for+neuronal+functioning%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li>
<li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+transfer+produces+a+predictable+cellular+phenotype%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li>
<li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis%3A+Application+of+an+excel-based+qPCR+preparation+software+%28PREXCEL-Q%29%5BTitle%5D)+AND+%22Int.+J.+Biomed.+Sci%22%5BJournal%5D)">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li>
<li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+capture+microdissection+in+forensic+research%3A+a+review%5BTitle%5D)+AND+%22Int+J+Legal+Med%22%5BJournal%5D)">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li>
<li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy%5BTitle%5D)+AND+%22Pharmacol.+Ther%22%5BJournal%5D)">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li>
</ol>

Dr. Eberwine is an inventor on the aRNA patent that has been licensed to LBS technologies in which Dr. Eberwine is a share-holder.

Notes and Troubleshooting
<ul>
<li>You may want to take before and after images showing that the target cell was isolated and that the remaining penumbra was left untouched.</li>
<li>If too much solution is entering the pipette as you are harvesting, you can use 3 way Luer-Lock fittings and the plunger for the syringe to hold positive pressure in the tubing. The desired volume will vary with the type of processing but up to 5 &mu;l should be fine for most applications.</li>
</ul>
General tips on molecular biological techniques
<ul>
<li>Vortex all stock tubes and spin them down before adding to a reaction. NEVER vortex enzymes.</li>
<li>Thoroughly mix reactions before adding the enzyme to ensure that the buffer is at the proper concentration.</li>
<li>Keep enzyme stocks at -20&deg;C if possible by using a stratacooler. Otherwise, remove right before use, keep on ice, and return immediately to -20&deg;C.</li>
<li>Always make master mixes when preparing more than one reaction to reduce pipetting errors. Calculate the volume required based on the number of samples and add 10-20%.</li>
<li>Store RNA at -80&deg;C to retard degradation. RNA is most stable stored in buffer to retard apurination of RNA that occurs in acidic conditions.</li>
</ul>
General Outline of the aRNA Procedure
Figure 4A illustrates the first round of the aRNA procedure. In the first strand reaction, the poly-T portion of the T7-oligo(dT) primer selects for mRNA species (long white rectangle) by binding to the polyA tails. Some microRNAs are also polyadenylated and will be captured by this procedure. More importantly, however, the most abundant RNAs in the cell, ribosomal RNAs, will not. This oligo acts as a primer for Reverse Transcriptase to synthesize a complementary strand of cDNA (long grey rectangle) using the mRNA as a template. The T7 portion of the T7-oligo(dT) primer incorporates the T7 RNA polymerase promoter in frame with the sequence antisense to the starting mRNA. This is used later in the in vitro transcription reaction.
Next, the mRNA in the mRNA/DNA hybrid created in the preceding step is partially hydrolyzed by Rnase H creating RNA "primers" (small white rectangles) similar to the Okazaki fragments created in lagging strand DNA synthesis. DNA Polymerase I uses the RNA fragments to prime DNA synthesis using the DNA complementary to the mRNA as a template. When it reaches the next RNA fragment, its 5' to 3' nuclease activity removes the ribonucleotides and replaces them with deoxyribonucleotides. DNA ligase is added to ligate any strands where the replacement of the leading strand is not complete. T4 DNA Polymerase is added to fill in the areas where RNA fragments served as initial primers for DNA Polymerase I creating a blunt-ended double stranded cDNA that is then purified before performing the IVT reaction.
In the IVT reaction, T7 RNA polymerase binds to the T7 promoter incorporated into the double stranded cDNA and synthesizes antisense RNA molecules (long black rectangles) using the sense strand as a template. This serves as the amplification step in which thousands of antisense RNA molecules are produced from each double stranded cDNA molecule (Figure 4A).
The second round, as depicted in Figure 4B, begins with a reverse transcription reaction that is slightly different from that of the first round since the starting RNA is antisense (solid black rectangle) and lacks the polyadenylated tail that was targeted by the T7-oligo(dT) primer in the first round. Therefore, this reaction is primed with random primers (small grey rectangles) and the RNA subsequently denatured. The second strand reaction is then primed by the T7-oligo(dT) primer, which binds to the poly-A sequence at the 3' end of the sense RNA created in the preceding reverse transcription reaction. Another IVT reaction is performed in the same manner as in the first round. This second round is usually repeated at least once to achieve three rounds of amplification from a single cell.
Applications
The techniques that we have presented in this article can be translated into a large number of applications. The single cell isolation protocol can be modified for use in acute slices.14 Although technically more challenging, the same principles apply in this alternate preparation. Additionally, if the size of the pipette is slightly adjusted, recordings of the physiology of the cells can be made before harvest allowing for a well-controlled investigation of molecular mechanisms behind physiological outputs. Another slight modification is to isolate processes from the cell soma.15 For this application, collect cell bodies with one pipette and then go back with a fresh pipette and collect 100-300 identified dendrites or axons per collection tube.16
Once cells have been harvested, comparisons of mRNA abundances and compositions can be made between different and even within the same cell populations. Incorporating biotinylated-UTP into the third round aRNA allows for microarray analysis to determine these relative mRNA abundances. The composition of the original mRNA population can also be determined after the aRNA procedure using next generation sequencing. The amplified aRNA can also be used to confirm cell phenotype conversion studies in which a full set of mRNAs from one cell type are transfected into a different cell type in order to induce transition of the phenotype of the latter cell type into that of the former, a procedure developed by the lab and known as TIPeR.17 These studies are particularly useful for studying disease states and cell phenotypes and such studies are currently ongoing in the lab. RT-PCR or qPCR can be performed on the amplified material to confirm the expression of cell-specific genes. Additionally, evaluations of the efficiency of transfection or transduction can be made at the single cell level.
Advantages and Limitations
As stated in the abstract, isolation of single cells for analysis eliminates the averaging effects seen with analysis of heterogeneous cell populations. These averaging effects misrepresent mRNA abundances within a single cell by over-representing abundant transcripts and averaging out and preventing detection of many low-abundance transcripts. Flow cytometry can be used to sort individual cells, but this method requires knowledge of cell specific markers and expensive equipment.18 Laser capture microdissection either with UV or IR laser capture microdissection systems allow for single cell and even subcellular capture but requires cells of interest to be located at the surface of very thin sections.19 Similar to flow cytometry, laser capture microdissection also requires expensive equipment.
One of the major benefits of the electrode based collection technique described above is that valuable electrophysiological data can be obtained from the cell of interest before harvest, allowing for functional and transcriptome analysis to be performed on the same cell.20 A downside of our technique is that it does require experience using micromanipulators. Investigators familiar with micromanipulators will find this technique very intuitive; however, individuals with no such experience will need to become comfortable with the fine movements required.
Inherent in any amplification technique is the preferential amplification of certain transcripts based on size and nucleotide composition.4,6,7 Polymerase chain reaction (PCR) based techniques such as reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) result in exponential amplification of transcripts, whereas the aRNA amplification procedure results in linear amplification. Thus, one of the major advantages of the aRNA procedure lies in its ability to better preserve the relative abundances of mRNA transcripts by only linearly amplifying any errors or biases that occur in the amplification process as opposed to exponentially amplifying said errors and biases. 
The aRNA procedure coupled with microarray analysis allows for comparison of mRNA abundances between single cells of similar or different morphology, treated or untreated. Additionally, amplified material can be submitted for next generation sequencing.5,8 However, care should be taken in such analyses since, in contrast to the use of random primers for the procedure, the oligo-dT primed procedure described above biases amplification of the 3' ends of mRNA and generally results in slight shortening of subsequent amplified material with each round. Care should be taken in analyzing microarray results with standard methods as some false absent calls can arise from slightly shortened amplified material. In addition, while sequencing results will indeed provide the full 5' sequence of most of the original mRNA, the 5' sequences of some mRNA might be missed. For these reasons, the number of rounds of amplifications should be limited.

A correction was made to <a href="http://http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

<table><tbody><tr><td>Spiegelgas coverslips</td><td>Carolina Biological</td><td>63-3029</td><td></td></tr><tr><td>Nunc 35x10 mm culture dishes</td><td>Fisher Scientific</td><td>12-565-90</td><td></td></tr><tr><td>Water for cell culture</td><td>Lonza Inc.</td><td>17-724Q</td><td>For making solutions used for cell culture and rinsing coverslips</td></tr><tr><td>Poly-D-Lysine MW70-150K</td><td>Sigma-Aldrich</td><td>6407</td><td></td></tr><tr><td>Laminin, ultrapure</td><td>BD Biosciences</td><td>354239</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B0252</td><td></td></tr><tr><td>MEM with Earle&amp;rsquo;s salts and glutamax</td><td>Invitrogen</td><td>41090-101</td><td>For plating cells</td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8769</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Horse serum</td><td>Invitrogen</td><td>16050</td><td></td></tr><tr><td>Cytosine beta-D-arabinofuranoside</td><td>Sigma-Aldrich</td><td>C1768</td><td></td></tr><tr><td>MEM with Earle&amp;rsquo;s salts and L-glutamine</td><td>Invitrogen</td><td>11095-098</td><td>For growing cells</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>B27 serum-free supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td><td>Kimble Chase</td><td>34500 99</td><td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14175</td><td>Any solution will work as long as the components won&amp;rsquo;t interfere with any future processing (i.e. no Ca&lt;sup&gt;2+&lt;/sup&gt; or Mg&lt;sup&gt;2+&lt;/sup&gt;)</td></tr><tr><td>1ml syringe</td><td>BD Biosciences</td><td>309628</td><td></td></tr><tr><td>Needle</td><td>BD Biosciences</td><td></td><td>Gauge depends on the diameter of the pipettes</td></tr><tr><td>dNTP mix</td><td>Amersham</td><td>28-4065-51</td><td></td></tr><tr><td>dt-T7 oligo</td><td>Custom</td><td>Midland Certified</td><td></td></tr><tr><td>Second strand buffer (5x)</td><td>Invitrogen</td><td>Y01129</td><td></td></tr><tr><td>DTT</td><td></td><td></td><td>Supplied with second strand buffer</td></tr><tr><td>E.coli DNA Ligase</td><td>Invitrogen</td><td>100002324</td><td></td></tr><tr><td>DNA polymerase I</td><td>Invitrogen</td><td>100004926</td><td></td></tr><tr><td>Rnase H</td><td>Invitrogen</td><td>18021-071</td><td></td></tr><tr><td>Megascript T7 kit</td><td>Ambion</td><td>AM1334</td><td></td></tr><tr><td>Random primers</td><td>BMB</td><td>11034731001</td><td></td></tr><tr><td>Superscript III Reverse Transcriptase</td><td>Invitrogen</td><td>56575</td><td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td></tr><tr><td>MEGAclear Kit</td><td>Ambion</td><td>1908</td><td></td></tr><tr><td>MinElute Reaction Cleanup Kit</td><td>Qiagen</td><td>28206</td><td></td></tr><tr><td>T4 DNA polymerase</td><td>Invitrogen</td><td>100004994</td><td></td></tr><tr><td>Rnasin</td><td>Promega Corp.</td><td>N251B</td><td></td></tr><tr><td>Illumina TotalPrep RNA Amplification Kit</td><td>Ambion</td><td>AMIL1791</td><td></td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instrument Co.</td><td>P-87</td><td>Sutter has many other models, many are discussed in the cookbook</td></tr><tr><td>Micromanipulator</td><td>Olympus Corporation</td><td></td><td>Many other micromanipulators will work such as the newer Eppendorf models</td></tr><tr><td>Pipette Holder</td><td>Warner Instruments</td><td>MP-S15A</td><td>Will vary with micromanipulator and pipette O.D.</td></tr><tr><td>Bioanalyzer RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr></tbody></table>

transcriptome analysis of single cells

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 ...

The overall goal of this procedure is to prepare samples from single cells for transcriptome analysis. This is accomplished by first finding a cell suitable for isolation. The micro pipette is then positioned next to the cell to be isolated.Next suction is applied to the syringe until the cell is pulled into the tip of the micro pipette. The final step of the procedure is to break the tip of the micro pipette into the collection tube and deject the remaining contents of the micro pipette into the tube. Ultimately, results can be obtained that show differences in mRNA populations between cells through microarray analysis of material amplified using the arna amplification method, also described in the protocol or for other applications such as PCR or next generation sequencing.Although this method can provide insight and the differences between single cells, it can also be applied to subcellular compartments such as dendrites or classes of dendrites, and provide important information into dendrite function. Demonstrating the procedure will be Jennifer Singh, a postdoc in my lab Prior to the start of this protocol, primary neurons from embryonic day 18, rat Hippo Campi were plated onto 12 millimeter cover slips in a 35 millimeter dish and maintained as described in the written protocol. To prepare pipettes for harvest, use a micro pipette puller to pull autoclave glass pipettes to a diameter appropriate for electrophysiology.Using the manufacturer's preset program, the bore size is not critical since the tip will be broken prior to cell collection. Carefully store the pipettes in a dust-free environment, such as a micro pipette storage jar, where the tips will remain intact when ready to harvest cells. Prepare the desired number of 1.7 milliliter einor tubes by adding two microliters of PBS for storage of the harvested material.Remove 1 35 millimeter dish of cover slips from the incubator in a tissue culture hood. Quickly transfer one cover slip from this dish to the lid of a new 35 millimeter dish containing HBSS. It is critical to return cover slips that are not being manipulated to the incubator as the cells become unhealthy.If left out for extended periods of time. An inverted light microscope with a 40 x objective fitted with a micro manipulator is necessary for cell harvesting. Use a pipette holder with flexible tubing leading away from the holder, affix the tubing to a stable surface to prevent unwanted movement of the pipette during collection.At the end of the tubing, insert a needle so that a one milliliter syringe with a lure lock connection can be attached and changed in between users. Prepare the micro pipette by breaking the tip in a controlled fashion by holding a Kim wipe taut between the fingers of one hand and very gently brushing the tip of the pipette across the paper one to two times. The opening should be approximately 75 to 100%of the target cell size.Secure the micro pipette in the micro pipette holder and affix the micro pipette holder to the insert on the micro manipulators. Finally, set the 40 x objective. Place the dish on the microscope stage and find a region of cells suitable for harvest.In the case of primary neuronal cultures, the cell should be relatively isolated from its neighbors. To prevent harvest of surrounding cells and or processes, place the cell to be harvested in the center of the field of view. Use the micro manipulator to advance the micro pipette toward the solution in the dish into the light path.Lower the pipette tip into the solution. Looking through the eyepiece, the pipette should appear as a shadow in the field of view. Apply positive pressure from this point on by blowing lightly through the syringe well above the plane of cells.Adjust the position of the pipette tip until it is within the field of view and focus on the pipette tip using the course focusing knobs. At this point, assess whether the bore size of the pipette is too large or too small. If the bore size is incorrect, replace with another micro pipette until the correct bore size is found To prevent pipette breakage in the collection region.Use the course focus to advance the focal plane before advancing the pipette tip towards the cells using the microm manipulators. As the cells become closer, use the fine focus until both the cells and the pipette tip are in focus. Stop applying positive pressure before the plaintiff cells is reached to avoid blowing them off of the cover slip.Position the pipette tip with the micro manipulators so that it is touching the cell soma to be harvested. Using the syringe, apply gentle suction by mouth until the cell enters the pipette tip. If the cell is not entering the pipette, gently move the tip closer to the cell and downward until it lifts from the cover slip.Use the micro manipulator to immediately move the pipette up and out of the solution. Remove the pipette from the holder. Hold the prepared 1.7 milliliter einor tube in one hand, and gently break the tip of the pipette on the side of the tube toward the bottom.Aiming for the 0.1 milliliter mark with the broken tip centered inside the tube, insert a needle attached to a one milliliter syringe into the top opening of the pipette. Rapidly depress the plunger, forcing the solution in the pipette to spray out into the tube. Take care not to touch the tip to any liquid on the sides of the tube.As capillary action will bring the liquid back into the pipette, the cell should remain in the very tip of the pipette, and this should be adequate to properly expel the cell. Quickly spin down the contents of the tube using a desktop micro fuge and freeze on dry ice or place on ice for further processing or for other applications such as PCR or next generation sequencing. Shown here is an example of a successful harvest of an isolated neuron.To begin, a relatively low density region of cells was selected. The pipette tip was then advanced toward the desired cell. Here the field of view is shown after the cell had been harvested.Note that the surrounding processes remain on the cover slip. Conversely, this figure demonstrates two images of pipette tips, which are an inappropriate size for effective harvesting. This tip will lead to incomplete harvest while the tip shown here will result in harvest of surrounding milieu.Following harvest, and A RNA amplification analysis, using a bioanalyzer is recommended to examine the distribution and quantity of amplified RNA. A successful amplification from single cell material will yield total amounts in the low micrograms and will have a distribution that is smooth and wide Once mastered, this technique can be completed in minutes if performed properly while performing this procedure, it's important to use RNA free technique.

Research

JoVE Journal

Neuroscience

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

Measuring the Kinetics of mRNA Transcription in Single Living Cells

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

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 Sequencing Analysis of Mouse Embryonic Cells

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

This paper presents methods for growing cardiac myocytes with different shapes, which represent different pathologies, and sorting these adherent cardiac myocytes based on their morphology at a single cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of heart failure.

An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Reusable Single Cell for Iterative Epigenomic Analyses

This article presents a protocol to perform comprehensive spatial transcriptomic profiling of host, viral, fungal, and microbiome RNA from formalin-fixed paraffin-embedded tissue sections using Stereo-seq, which enables high-resolution mapping of diverse transcriptomes while preserving tissue architecture.

Comprehensive Spatial Profiling of Species-agnostic Transcriptomes via Stereo-seq

Learn single-cell transcriptome analysis using aRNA amplification for neuroscience research with rat primary neuronal cultures. This method enables detailed study of gene expression variability between individual neurons.

Transcriptome Analysis of Single Cells

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