The authors would like to acknowledge the technical assistance of C. Zambataro during the filming of this protocol. We gratefully acknowledge the support of the Glenn Foundation for Medical Research (S.M.), The Hillblom foundation, and the National Institutes of Health for a Nathan Shock Center award (P30AG025708) and PO1AG025901. J.M.F. was supported by T32AG000266 awarded to the Buck Institute for Research on Aging.

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Free access and production of this of this article is sponsored Sigma-Aldrich.

This method has the possibility to generate cardiomyocytes for a number of functional as well as gene expression studies. The examination of single cells is a burgeoning area in gene expression analysis. The advantage of examining transcriptional levels at the level of the single cell is that it allows the examination of a pure cell population, which is not possible from whole tissue preparations. Additionally, single cell analysis allows for the examination of stochastic variation of mRNA levels in individual cells to define cell populations that were previously thought to be homogeneous 8,9. In addition to identifying genes which are potentially stochastic in their gene expression 10,11,12, the method also allows for the identification of rare cell populations defined by their gene expression profiles.
	While this method provides a number of exciting potential uses in gene expression analysis, there are some caveats and considerations in the use of single cells. The primary limitation to single cell analysis, is the collection of enough cells in the experiment to achieve statistical significance with regards to the metric of interest, for example variance. In cases where the numbers of cells isolated from the tissue of interest is not a limitation, one can examine hundreds of cells per group, using approaches such as next-generation sequencing, or nanofluidic arrays. However, in some cases, there may be difficulty in obtaining sufficient cells from the tissue of interest. This can in part be caused by idiosyncratic time intensive procedures for the collection of the targeted cell type. However, careful planning and preparation prior to proceeding with single cell collection may still allow for rigorous single cell analysis with limited technical error. When examining the results it must be considered, that even though this procedure for isolating cells has been used in numerous studies (including microscopy, electrophysiology, etc.), the isolation itself could potentially effect gene expression. As with any method which manipulates biological samples, the results of your findings should be carefully validated to ensure the single cell expression is representative of the tissue itself and not technical bias. Methods such as in situ hybridization may prove useful to verify these results in the intact tissue. Lastly, it is critical to ensure that the data generated is carefully checked for quality control. The data shown in Figure 8 demonstrates that qPCR assays may be very robust in their specificity, such as assay #13 (A13) or have a high level of variability which can lead to technical variance such as assay #34 (A34).

<table border="1">
<tbody>
 <tr>
 <td>Name 				of the reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>71378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Sigma-Aldrich</td>
 <td>60128</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pyruvic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>P4562</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>Sigma-Aldrich</td>
 <td>54457</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>Sigma-Aldrich</td>
 <td>M1020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaH2PO4 				monobasic</td>
 <td>Sigma-Aldrich</td>
 <td>P9791</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextrose</td>
 <td>Sigma-Aldrich</td>
 <td>G6721</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Sigma-Aldrich</td>
 <td>72068</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>O3777</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>Sigma-Aldrich</td>
 <td>21115</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protease, Type XIV</td>
 <td>Sigma-Aldrich</td>
 <td>P5147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase, Type 				I</td>
 <td>Worthington</td>
 <td>C9891</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1x DNA Suspension 				Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA)</td>
 <td>TEKnova, </td>
 <td>PN 				T0221</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma-Aldrich</td>
 <td>B8667</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium 				Pentobarbital</td>
 <td>Henry Schein Vet. 				Supply*</td>
 <td>Contact 				supplier for ordering</td>
 <td>&nbsp;*must be 				procured through a Veterinarian or lab animal professional</td>
 </tr>
 <tr>
 <td>ExoSAP IT</td>
 <td>&nbsp;Affymetrix/USB</td>
 <td>78201&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellsDirect 2x Rxn 				Mix</td>
 <td>Invitrogen</td>
 <td>11737-030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SuperScript III RT 				Platinum Taq Mix</td>
 <td>Invitrogen</td>
 <td>10928-042</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RT-STA Primer Mix</td>
 <td>IDT</td>
 <td>Custom</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nuclease Free 				water</td>
 <td>Sigma-Aldrich</td>
 <td>W4502</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>WTA2</td>
 <td>Sigma-Aldrich</td>
 <td>WTA2-50rxn</td>
 <td>&nbsp;</td>
 </tr>
<tr>
 <td>Qiaquick PCR 				purification kit</td>
 <td>Qiagen</td>
 <td>28104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Qiacube</td>
 <td>Qiagen</td>
 <td>9001292</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NanoDrop 				Spectrophotometer</td>
 <td>NanoDrop</td>
 <td>2000c</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BioAnalyzer DNA 				7500 kit</td>
 <td>Agilent</td>
 <td>7500 				kit</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>One Color Labeling 				kit</td>
 <td>Roche-NimbleGen</td>
 <td>5223555001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mus Musculus 				12x135k array</td>
 <td>Roche-NimbleGen</td>
 <td>5543797001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GenePix 4200A 				Scanner</td>
 <td>Molecular Devices</td>
 <td>4200A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TransPlex 				Complete Whole Transcriptome Amplification Kit (WTA2 Kit)</td>
 <td>Sigma-Aldrich</td>
 <td>WTA2-50RXN</td>
 <td>&nbsp; </td>
 </tr>
 </tbody>
</table>

single cell transcriptional profiling of adult mouse cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Watch this Scientific Journal Video about Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes at JoVE.com

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

single-cell-transcriptional-profiling-of-adult-mouse-cardiomyocytes

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Biology

17.2K Views.  Buck Institute for Research on Aging. Singoli profili di espressione delle cellule consente la dettagliata analisi di espressione genica di cellule singole. Noi descrivere i metodi per l'isolamento dei cardiomiociti, e preparare il lisati risultante sia per microarray intero trascrittoma o qPCR di obiettivi specifici.