1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington
Flynn, J. M., Santana, L. F., Melov, S. Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes. J. Vis. Exp. (58), e3302, doi:10.3791/3302 (2011).
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.
1. Step 1 - Cardiomyocyte isolation
|Final Conc.||g/L||g/500 mL||10x solution g/L|
|Pyruvic acid||3 nM||0.33||0.165||3.30|
Digestion Buffer A (Make three aliquots of this solutions)
Digestion Buffer B (Make one of these)
Collection Buffer (Make one of these)
Neutralization Wash Buffer (Make one of these)
** Depending on the source and batch of collagenase, the enzyme activity may vary. It may be necessary to optimize the amount of enzyme added to this buffer to prevent over-digestion.
2. Step 2 - Isolation of single cells
3. Step 3A - Single cell qPCR gene expression
To perform qPCR on single cells employ a protocol adapted from one developed for single cell gene expression by Fluidigm Corporation for their Biomark Nanofluidic qPCR arrays (Fluidigm - Advance Development Protocol #30)3,4.
|Reagent||Volume in μL (per reaction)|
|CellsDirect 2x Reaction mix||5.0|
|SuperScript III RT Plantinum Taq mix||0.2|
|RT-STA Primer mix (200 nM each assay)||2.5|
|Nuclease Free H2O (or 1x DNA suspension buffer)||1.3|
3. Step 3B - Whole transcriptome amplification and microarray of single cells
Extraction and Amplification
Labeling and NimbleGen Gene Expression Arrays
If the heart perfusion worked well there should be a high percentage of healthy heart cells that retain their typical rectangular morphology upon isolation. If the perfusion did not proceed well then there will be a large percentage of dead cells (see images in Figure 5). If the cells are correctly amplified using either the WTA2 or RT-STA methods then the end products should pass subsequent quality control tests for quality of mRNA samples by NanoDrop and BioAnalyser (Figure 6). For the microarray workflow, this is completed after WTA amplification where the mRNA is tested by both NanoDrop and Bioanalyzer. Representative positive results for this analysis are shown in Figure 6. The WTA2 samples should show a robust amplification with both the NanoDrop spectrophotometer reading, as well as the electropherogram readout from the Bioanalyzer (BA) chip (Figure 6). A table of genes which were stably detected via microarray of single cells is included as an example (Table S1). For the qPCR process, the quality of the data can be assessed by performing a melt step at the end of the PCR reaction to ensure that the primer sets are amplifying the desired product (Figure 7). If correct, this melt step should generate one specific melt peak. To illustrate this point, a subset of nanofluidic qPCR data is shown in a heatmap of peak melt temperature (Figure 8) 8. It is possible to assess the quality of a PCR product by its melt temperature to ensure the absence of non-specific products 5. Each row of this map represents one cell sample (S1-S40) tested in 43 separate qPCR assays (A1-A43). In this figure, it is clear that some assays are quite variable and are probably less reliable than the more stable assays with higher PCR specificity.
Figure 1. Overview of the single cell isolation and gene expression analysis. The procedure will demonstrate the surgical removal of the mouse heart and the isolation of individual cardiomyocytes. The methods discussed in this procedure include the methods for either qPCR or microarray analysis after whole transcriptome amplification (WTA). The WTA procedure begins with the lysis (A) then the binding of Sigma's universal primers (B) to the mRNA pool. The extension of these primers (C) and the amplification (D) phase create micrograms of amplified material. This amplification is then repeated (E) to generate enough material for the microarray procedure.
Figure 2. Representation of the location of the incisions to remove the heart from the mouse. Cut along the lateral wall of the rib cage (A), then cut along the inferior margin of the rib cage (B). Cutting the vessels above and below the heart allow for removal while still leaving enough of the aorta to cannulate the heart. Images adapted from M.J. Cook 6.
Figure 3. Diagram of the thorax of the mouse. The dotted lines indicate the locations of the incisions (A) for excising the heart from the thoracic cavity without damaging the heart tissue. Images adapted from MJ Cook 6.
Figure 4. Image of the aortic arch and its branches. The gray line indicates the ideal location of where to trim the aorta (A). The remaining aorta attached to the heart is cannulated so that the tip of the cannula (B) goes to the appropriate level within the aorta (C). Images adapted from F. Gaillard 7.
Figure 5. Selection of single cells for gene expression analysis. Each panel shows cardiomyocytes imaged under the light microscope showing typical morphology of cardiomyocytes. Healthy cardiomyocytes are indicated by the green arrows. Cells which are dead or dying are shown with red arrows. These dead cells should not be used in the analysis.
Figure 6. Quality control WTA results for the single cell amplification. (A) Image of a NanoDrop spectrophotometer readout that is appropriate for the amplified transcripts from the WTA reaction. (B) The readout from BioAnalyzer chip shows the results from three amplified cells.
Figure 7. qPCR amplification curves (Left chart) and peak melt temperature curves (Right chart). (A) The expression results from a quality assay are shown with a single melt peak despite different amplification curves. (B) Melt curves for a poor primer set, which has highly variable melt peaks which is not ideal for expression analysis.
Figure 8. Quality control results for the single cell qPCR reactions. This heat map was created to display the melt temperatures as a color scale. The peak melting temperature for each qPCR assay (A1-A43) is shown for 40 individual cells (S1-40). The assays were clustered according their melt temperature. By looking down the column for each assay it is possible to see the variation melt across all the samples. This figure demonstrates that some qPCR assays are very specific while others are highly variable and thus are not suitable for single cell analysis.
Table S1. Supplemental table of microarray data. These genes are listed according to the robustness in which they are detected in single cardiomyocytes across a large dataset. This gene list indicates the sensitivity of detection for a number of genes that are typically expressed in single cardiomyocytes.
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).
Free access and production of this of this article is sponsored Sigma-Aldrich.
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.
|Protease, Type XIV||Sigma-Aldrich||P5147|
|Collagenase, Type I||Worthington Biochemical||C9891|
|1x DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA)||TEKnova, Inc.||PN T0221|
|Sodium Pentobarbital||Henry Schein||Contact supplier for ordering||*must be procured through a Veterinarian or lab animal professional|
|CellsDirect 2x Rxn Mix||Invitrogen||11737-030|
|SuperScript III RT Platinum Taq Mix||Invitrogen||10928-042|
|RT-STA Primer Mix||IDT||Custom|
|Nuclease Free water||Sigma-Aldrich||W4502|
|Qiaquick PCR purification kit||Qiagen||28104|
|BioAnalyzer DNA 7500 kit||Agilent Technologies||7500 kit|
|One Color Labeling kit||Roche Group||5223555001|
|Mus Musculus 12x135k array||Roche Group||5543797001|
|GenePix 4200A Scanner||Molecular Devices||4200A|
|TransPlex Complete Whole Transcriptome Amplification Kit (WTA2 Kit)||Sigma-Aldrich||WTA2-50RXN|