Name: target cell pre-enrichment and whole genome amplification for single cell downstream characterization
Uploaded: 2018-05-15T12:30:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization at JoVE.com

This study was funded by the Austrian Science Fund (FWF), project-no. I 1220-B19 (to P.S.) as part of the ERANET project in Translational Cancer Research (TRANSCAN) &#34;Circulating Tumour Cells as Biomarker for Minimal Residual Disease in Prostate Cancer (CTC-SCAN)&#34;. PhD candidate S.C. was trained within the frame of the PhD Program Molecular Medicine of the Medical University of Graz. The authors gratefully acknowledge Nina Schl&#246;gl, Daniel Kummer, Gabi Wendt, Claudia Chudak, Julia Schulz, and Johanna Schiller for their expert technical assistance. The authors thank Georg Peinhaupt for graphical design support.

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The authors have nothing to disclose.

The described protocol aims at retrieving cells from a functionalized wire (referred to as Device) for ex vivo single-cell genomic analysis. We tested three EpCAM-positive cell lines (HT-29, LNCaP, and VCaP), but in principle, this method can be applied to any cell line expressing EpCAM. EpCAM-positive target cells can be presented to the Device as pure cell suspension (see 2.1.) or mixed into a surplus of background cells (e.g. peripheral blood, see 2.2.). Whereas especially the latter might be used to test isolation capacities under rare cell conditions, fine tuning of the number of cells attached to the wire can be done using the described low-volume approach (see 2.3.). As differences between cell lines are to be expected, suitable cell densities for charging the wire, rotation speed as well as incubation time need to be tested empirically by evaluating the number of attached cells using an immunofluorescence microscope.
For genomic downstream applications at the single-cell level WGA, is required. The WGA method of choice may differ between labs and, in principle, our method is open to all methods than can handle samples obtained from micromanipulation or laser microdissection. In this protocol, we use a method based on enzymatically fragmented DNA due to a better performance as compared to a heat-fragmentation based WGA7. Optional, single cells can be forwarded to isothermal WGA allowing subsequent DNA profiling20,21.
The described protocol aims at recovering intact cells after being attached to a new generation of functionalized wires for genomic single-cell analysis. The first generation of functionalized wires, which also represent the only CE-approved in vivo enrichment devices on the market so far, were used for enumerating CTCs in metastasized cancer patients. Previous publications and our own data suggest the in vivo isolation technique to be more sensitive compared to the current gold standard technology2. Thus, equipping this in vivo isolation technique with a recovery feature as realized in the Device allows combining high CTC recovery with characterization at the single-cell level.
However, the Device is not cleared for the use in patients, thus, clinical data are not available. Ex vivo applications (i.e. isolating CTCs from peripheral blood after sampling) are not recommended as the technology is adapted to the settings present in the cubital vain, e.g. low diameter of the vain (increasing the probability of direct contact between CTCs and catching antibodies) and long retention time (30 min, allowing 2-3 L of blood to pass the wire). However, as outlined in section 2.2. target cells can also be isolated from mixed samples7.
A current limitation of the method is the inefficient recovery of unfixed cells after detachment from the wires. This, however, might be improved by a cell fixation step prior to the detachment treatment.
In summary, this protocol is a first step towards the combined use of an in vivo isolation technique with cell recovery for genetically characterizing single cells. Further efforts need to address the optimization of the cell recovery and clearance for its application in patients1,2. However, personalized medicine for treatment monitoring and therapy decisions is beyond enumeration of CTCs. Thus, this method is a first step towards genomic CTC characterization at the single cell level.
Applications towards single-cell transcriptome analysis seem feasible as intact cells are harvested. However, it remains to be assessed whether the recovering procedure has an impact on RNA integrity (e.g. forwarding recovered single cells to direct lysis followed by RT-qPCR22). If successful, characterization of single cells (e.g. CTCs) can be shifted to the transcriptome level , allowing more detailed analyses. In this regard, RNA-seq using Smart-seq2 provides a valuable tool for RNA downstream analysis of single cells as the preamplified cDNA (obtained during RNA-seq library preparation) can be subjected to quantitative PCR. This will allow a combined target and screening-based analysis of single recovered cells22,23.
The current functionalized wires are based on anti-EpCAM antibodies which is a widely used epithelial marker for positive selection of CTCs24. As several CTCs might downregulate epithelial markers such as cytokeratins or EpCAM25, adding antibodies such as HER2/new would increase the chance of CTC isolation. Several antibody based enrichment strategies have been developed and reviewed by Ferreira et al. in 201626. A mixture of antibodies immobilized on a wire could be a future improvement of the technology leading to the isolation of a higher number and additional subtypes of CTCs.
It is mandatory for all steps involving wire handling to keep the functional part of the wire submerged in solution in order to maintain its functionality. We recommend to place the wire in 1x PBS during evaluation (microscope; e.g. steps 3.1. - 3.3.) and storage. In order to avoid inappropriate staining, we recommend using freshly prepared staining solution in steps 1.2.1. and 1.2.2. Weakly stained cells hamper cell counting on the wire and may lead to the underestimation of the attached cells.
It is necessary to avoid plugging the Pasteur pipette with the rubber cap when inserting the wire in the Pasteur pipette for subsequent loading of the cell suspension (step 2.3.4.). The rubber cap is used to hold the wire in place so that the functional part is situated inside the tip of the Pasteur pipette. If the cap is attached too firmly to the rear of the Pasteur pipette, loading of the cell suspension is not possible. In this case, ease the cap such that the air which is displaced by the loaded cell suspension can leave the pipette.
Bending the wire is crucial for its orientation during the cell counting in steps 3.2. and 3.3. Mount the wires using the adhesive tape such that the non-functional end of the wire comes to lie to one side. After scanning of the entire length of the functional part, the wire is rolled 180&#176; so the other half of the functional wire can be scanned. This step is important for initial experiments to assess the number of cells on the wire. Once the number of cells for a certain cell line and procedure is evaluated, the procedure can be repeated without cell counting (e.g. only checking for presence of cells or omitting this step). Careful pipetting is crucial to avoid cell loss when reducing the volume of the supernatant in step 4.8. Make sure pipetting is done smoothly without causing turbulences to the pellet.

Recently, CellCollector DC01, a medical wire functionalized with anti-EpCAM antibodies for isolating CTCs from peripheral blood of cancer patients, was added to the methodical spectrum of CTC enumeration1,2,3. In a currently ongoing study in non-metastatic prostate cancer, this functionalized wire reports almost twice as many patients to be CTC-positive and higher CTC counts in CTC-positive patients as compared to CellSearch, the gold standard in CTC enumeration3. Due to this encouraging performance, isolation of cells from a functionalized medical wire for single-cell analysis would be desirable, but neither enzymatic treatment with cell detachment solutions (e.g. trypsin) nor laser microdissection allows the recovery of intact cells (data not shown).
To allow the detachment of captured cells, a new generation of functionalized wires was equipped with a specific polymer. This polymer, which links the capture antibodies to the wire, is susceptible to a release buffer treatment allowing detachment of intact cells (CellCollector DC03 referred to as Device). The new functionalized device, allows isolation of target cells from various concentrations of cancer cell line cells spiked into bovine serum albumin (BSA)/phosphate buffered saline (PBS) and peripheral blood, respectively.
To ease the visual detection of cells on the Device and after recovery, the target cancer cells are labelled with carboxyfluorescein succinimidyl ester (CFSE) and a DNA stain before the recovering treatment (i.e. charging and detaching). The treatment&#39;s effects on DNA quality of single cells were previously evaluated after WGA by means of a quality control PCR4,5, array-CGH6,7&#160;and targeted sequencing7 showing no difference compared to non-treated cells micromanipulated from cell suspensions7. The advantage of this method lies in the combination of rare target-cell pre-enrichment and the recovery of cells for single-cell downstream analysis (Figure 1). The current CE-labelled in vivo collection device is generally used for enumeration of CTCs rather than for single-cell molecular characterization2,8. However, more comprehensive analysis to investigate heterogeneity among CTCs long for analysis at the individual cell level (i.e. targeted sequencing at the single-cell level). Other cell-based methods are based on immunomagnetic isolation of EpCAM-positive CTCs and single-cell handling based on dielectrophoresis for subsequent molecular genetic analysis9,10. Molecular characterization of CTCs is an important requirement for their useful implementation in a clinical setting and is equally important in basic research of the metastatic cascade. In parallel to CTCs, circulating tumor DNA (ctDNA) has become of great importance as it allows DNA analysis of the tumor burden with minimal technical isolation procedures11,12. The cell based approaches might serve as a complementary contribution as it allows for RNA13,14 and protein15 expression analysis and also for CTC derived cell cultures or xenografts16,17. Although obstacles such as low cell recovery and clearance for the use in patients still need to be overcome, the catch and release method takes an important next step towards characterization of rare target cells.

<table>
	<tbody>
		<tr>
			<td>Gilupi Release Buffer</td>
			<td>Gilupi</td>
			<td>GIL083</td>
			<td>Used to detach cells from the wire</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A Medium (1x) suppl. with L-Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>16600-082</td>
			<td>Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1 M)</td>
			<td>PAA</td>
			<td>S11-001</td>
			<td>Supplement for McCoy&#39;s 5A Medium</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum Gold</td>
			<td>PAA</td>
			<td>A15-151</td>
			<td>Supplement for McCoy&#39;s 5a Modified Medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Biowest</td>
			<td>L0018-100</td>
			<td>Supplement for McCoy&#39;s 5a Modified Medium</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1x PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Biowest</td>
			<td>L0950-100</td>
			<td>Cell detachment solution (cell culture)</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>Type 1003</td>
			<td>Used for prewarming solutions</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Used to incubate cells (cell culture)</td>
		</tr>
		<tr>
			<td>50 mL reaction tubes</td>
			<td>VWR</td>
			<td>525-0403</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller mixer</td>
			<td>Stuart Scientific</td>
			<td>SRT6D</td>
			<td>Used to attach cells to the wire while keeping the cells from sedimenting</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td>Used to label living cells (cytoplasmic label)</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>H3570</td>
			<td>Thermo Fisher Scientific</td>
			<td>Used to stain DNA (nucleic counterstain)</td>
		</tr>
		<tr>
			<td>Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Heraeus Megafuge 40R</td>
			<td>Used for pelleting cells</td>
		</tr>
		<tr>
			<td>Observer Z1 (Fluorescence/laser microdissection microscope)</td>
			<td>Carl Zeiss Microimaging</td>
			<td></td>
			<td>Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td>Used for coating glass/plastic surfaces</td>
		</tr>
		<tr>
			<td>MS1 Minishaker</td>
			<td>Sigma-Aldrich</td>
			<td>Z404039</td>
			<td>Used for vortexing</td>
		</tr>
		<tr>
			<td>Vacutainer EDTA (or Na-heparin) tubes</td>
			<td>BD</td>
			<td>366450 (or 366480)</td>
			<td>Used as reaction tube for ex vivo capture of target cells</td>
		</tr>
		<tr>
			<td>Detektor CANCER03 DC03</td>
			<td>Gilupi</td>
			<td>GIL003</td>
			<td>Functionalized wire capable of isolating and detaching EpCAM-positive cells (also referred to as catch&#38;release, C&#38;R)</td>
		</tr>
		<tr>
			<td>Syringe needles (G20)</td>
			<td>VWR</td>
			<td>613-0554</td>
			<td>Used to puncture rubber caps in order to allow the non-functional part of the C&#38;R to lead through it</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Roth</td>
			<td>T729.1</td>
			<td>Used to count cells</td>
		</tr>
		<tr>
			<td>Pasteur pipettes 150 mm</td>
			<td>Volac</td>
			<td>D810</td>
			<td>Used for the low-volume application of cells to the C&#38;R</td>
		</tr>
		<tr>
			<td>Grease pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td>Used on glass slides to hold cell suspension in place</td>
		</tr>
		<tr>
			<td>Steril syringe filter (0.2 &#181;m)</td>
			<td>Corning</td>
			<td>431219</td>
			<td>For filtering freshly prepared Gilupi Release Buffer</td>
		</tr>
		<tr>
			<td>Delfia PlateShaker</td>
			<td>Perkin Elmer</td>
			<td>1296-003</td>
			<td>Used for agitation during cell detachment</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>30,125,150</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampli1 WGA Kit</td>
			<td>Silicon Biosystems</td>
			<td></td>
			<td>To be ordered via Silicon Biosystems</td>
		</tr>
		<tr>
			<td>Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario</td>
			<td>Zeiss/Eppendorf</td>
			<td></td>
			<td>Use micromanipulation at hand</td>
		</tr>
		<tr>
			<td>Microcapillaries (25 &#181;m in diameter), CustomTip Type I</td>
			<td>Eppendorf</td>
			<td>930001201</td>
			<td>Used for micromanipulating cells</td>
		</tr>
		<tr>
			<td>0.2 mL PCR tubes</td>
			<td>Biozym</td>
			<td>710920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytocentrifuge and equippment</td>
			<td>Hettich</td>
			<td>Universal 32</td>
			<td>Use cytocentrifuge at hand</td>
		</tr>
		<tr>
			<td>Thermo cycler</td>
			<td>Bio-Rad</td>
			<td>DNA Engine Dyad</td>
			<td>Every thermo cycler with heated lid can be used</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Roth</td>
			<td>CX73.1</td>
			<td>Use desk-top centrifuge at hand</td>
		</tr>
		<tr>
			<td>MembraneSlide 1.0 PET</td>
			<td>Zeiss</td>
			<td>415101-4401-050</td>
			<td>Membrane-coated glass slides used for laser microdissection</td>
		</tr>
		<tr>
			<td>UV Stratalinker 1800</td>
			<td>Stratagene</td>
			<td>400072</td>
			<td>Use DNA cross linker at hand</td>
		</tr>
		<tr>
			<td>Standard PCR reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6x DNA Loading</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0611</td>
			<td>Use gel loading dye at hand</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>BioLabs</td>
			<td>N0551G</td>
			<td>Use 100 bp ladder at hand</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>840004</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electorphoresis station</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Use electrophoresis system at hand</td>
		</tr>
		<tr>
			<td>Gel Red Nucleic Acid Stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>Intercalating dye for DNA visualization In agarose gels</td>
		</tr>
	</tbody>
</table>

target cell pre-enrichment and whole genome amplification for single cell downstream characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

Cells after CFSE and nucleic staining can be evaluated using an immunofluorescence microscope equipped with filters for DAPI and FITC. Nuclei present with a bright counterstain in the DAPI channel whereas cytoplasm of the cells show uniform labelling with CFSE with heterogeneous inter-cell intensity (Figure 2A and 2E). Similar shape and staining intensities are expected from cells attached to the wire (Figure 2B and 2F) as well as detached from the wire and recovered on a slide (Figure 2D and 2H).
High cell densities (e.g. &#62;1 000 000 cells/mL) may result in total coverage of the wire with cells when charging the wires. However, lower cell densities, changes in rotation speed during incubation, and the use of different cell lines affect the isolation efficiency and need to be tested separately. When the wires were incubated with 5 mL of HT-29 cells at 100 000 cells/ mL as described in section 2.1., a mean of 254 &#177; 103 cells (n = 5) were captured on the wires. With the same experiment using LNCaP cells, a mean of 440 &#177; 319 cells (n = 5) per wire was obtained.
Presenting 500, 5 000, and 50 000 HT-29 cells in a background of 5 mL of peripheral blood to wires (according to protocol section 2.2.) resulted in the capture of 75 &#177; 18 cells, 25 &#177; 9 cells, and 47 &#177; 57 cells (n = 3, each), respectively. If the wire is charged with 15 &#181;L of cell suspension (1 000 000 cells/mL) according to the protocol in section 2.3., up to 1 000 (HT-29) cells may attach to a wire.
Cell detachment efficiencies ranged from 81% in HT-29 cells (range 54.9% - 93.2%, and n = 5) to 81% (range 63.51%-92.87%, n = 6) and 91% (range 84.7% - 95.3%, n = 6) in LNCaP and VCaP cells, respectively. Similarly, the recovery of detached cells differs between cell lines: A mean of 28% of detached HT-29 cells were recovered by cytocentrifugation. Whereas the recovery rate for LNCaP was lower than 10%, the mean recovery rate for VCaP cells was 55%.
Approximately 75% of single cells subjected to WGA yield high quality WGA products: This is 1) smear of WGA products ranging from 0.1 to &#62; 1 kb (Figure 4, top panel) and 2) three or four bands in the 4plex quality control PCR (Figure 4, bottom panel). WGA products can be used for 1) array-CGH profiling meeting the quality criteria for single-cell array-CGH profiles 6,7 and 2) targeted NGS after re-amplification WGA 7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56394/56394fig1.jpg" /> 
Figure 1: Workflow for isolating and recovering target cells for single-cell analysis. (A) Cells are cultured to 90% confluency and (B) harvested to obtain a single-cell suspension. After staining with CFSE and nucleic stain, (C) the functionalized wire is incubated in the presence of the cells either in a volume of 5 mL using reaction tubes or in a low volume using a Pasteur pipette tip. The latter allows for application of cell suspension volumes as low as 15 &#181;L. (D) Cells need to be detached within 4 h after charging. Therefore, the wire is placed into a 1.5 mL reaction tube filled with release buffer. After 15 min incubation on a rocker and 10 min centrifugation, the wire is removed and the cell suspension is centrifuged to pellet the cells. (E) Recovered cells are either transferred to a glass slide for single-cell micromanipulation (top) or cytocentrifuged onto a membrane-coated slide and forwarded to laser microdissection (bottom). (F) Finally, harvested single cells are amplified by means of whole genome amplification (WGA). WGA products are compatible with array-CGH and targeted NGS downstream analysis.EpCAM epitope: green circle on cell surface. Functionalized wire: Device, grey triple-helical wires. Polymer: purple lines. Anti-EpCAM antibodies: orange. Release buffer activity: red arrow. Grease marker to hold cell suspension in place: dark blue ellipse. Recovered cell suspension: light blue. Capillary for micromanipulation: black line. Magnification: recovered cell harvested by micromanipulation, cytospin on membrane-covered slide containing recovered cells (grey filled ellipse). Magnification: recovered cell being laser microdissected (grey circular line: membrane from membrane slide serving as backbone for laser microdissection), objective with laser beam (blue line approaching the membrane slide from below). <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56394/56394fig2.jpg" /> 
Figure 2: Visualizing labelled cells. (A,E) Cells before charging: Cells are labelled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity. (B,F) Labelled cells captured on the wire. (C,G) Wire after cell-detachment procedure. (D,H) Cells detached from wire and recovered by cytocentrifugation onto a slide. CFSE: FITC channel (green). DNA stain: DAPI channel (blue). <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56394/56394fig3.jpg" /> 
Figure 3: Wire and storage compartment. (A) Compartments of the wire. (B) Detail of the functionalized part. <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56394/56394fig4.jpg" /> 
Figure 4: Quality control of WGA products obtained from single micromanipulated cells. Top panel: WGA products obtained from single cells typically result in DNA smears ranging from 0.2 kb to &#62; 1.0 kb with a peak intensity at around 0.5 kb. Samples number 1, 7 and 13 (indicated with asterisks) show suboptimal or no DNA smears. Bottom panel: WGA products are of sufficient quality if the 4plex PCR yields three or four of four PCR products (at 100, 200, 300, and 400 bp). Samples 1, 7, 10, and 13 show fewer than three bands and would be excluded from further analysis. Lane M contains a 100 bp ladder and asterisks indicate the samples of insufficient WGA product quality. <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer sequence</td>
			<td>NOTE</td>
		</tr>
		<tr>
			<td>5&#8242;-gtt cca ata tga ttc cac cc-3&#8242;</td>
			<td>100 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-ctc ctg gaa gat ggt gat gg-3&#8242;</td>
			<td>100 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-agg tgg agc gag gct agc-3&#8242;</td>
			<td>200 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-ttt tgc ggt gga aat gtc ct-3&#8242;</td>
			<td>200 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-agg tga gac att ctt gct gg-3&#8242;</td>
			<td>300 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-tcc act aac cag tca gcg tc-3&#8242;</td>
			<td>300 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-aca gtc cat gcc atc act gc-3&#8242;</td>
			<td>400 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-gct tga caa agt ggt cgt tg-3&#8242;</td>
			<td>400 bp reverse primer</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences for 4plex quality control PCR

Watch this Scientific Journal Video about Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization at JoVE.com

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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