QIAscout

Highly viable single-cell isolation

Cells remain viable for several days and form subpopulations, irrespective of whether the pierced microrafts containing single cells are transferred to a cell culture dish or single cells present on microrafts are left intact and allowed to grow on the QIAscout array itself (see corresponding Application Note titled “Rapid and Efficient Method of Isolation and Recovery of Highly Viable Single Cells Free of Contamination” under the “Product Resources” tab).

Highly pure single-cell isolation

The release needle and the magnetic wand do not act as sources of contamination while isolating single cells using the QIAscout. This was verified using two approaches, qPCR (see Figure “No DNA contamination observed with the release needle in qPCR analysis”) and Pyrosequencing (see Figure “No cell contamination observed with the magnetic wand in Pyrosequencing analysis”), after processing cells using the REPLI-g Single Cell Kit.

Highly targeted single-cell isolation

The QIAscout method allows efficient and reliable isolation of single targeted cells. Selection of cells can be based on different cellular parameters like cell morphology or fluorescence. Conventional manual methods like pipetting and serial dilution are less successful in selective isolation of single cells when compared to the novel QIAscout technology (data not shown).

Unique miRNA expression profiling from single cells

The precise and affordable single-cell isolation technology in combination with miScript Single Cell qPCR Kit and miScript miRNA PCR Array facilitates the downstream expression analysis of miRNAs from individual cells. In bulk cell analysis “cellular averages” mask intrinsic transcriptional variability across individual cell subpopulations. Distinct cell populations can be effectively revealed by single-cell analysis using the QIAscout for miRNA expression profiling (see Figure “Principle Component Analysis (PCA) of miRNA expression of single cells”).

The QIAscout system is an effective and fast method to isolate viable and pure single cells, ensuring minimal manipulation of the cellular status. The core technology of the QIAscout is the QIAscout array, which is composed of 12,000 individual microrafts. Each microraft is 200 µm in length and 200 µm in width and is securely held within a micron-scale well of the QIAscout array. Within the QIAscout array, the microrafts serve as releasable culture sites for individual cells or colonies.

The QIAscout array is ideal for various cell types including adherent cells, suspension cells, cell lines, primary cells, and fluorescent cells providing an environment suitable for their growth and viability similar to any standard cell culture dish. QIAscout arrays are mounted on inverted microscopes and can be imaged by brightfield, fluorescence and confocal microscopy.

A motorized release device is mounted onto a 4x, 5x or 10x objective of common inverted lab microscopes to allow for selection and picking of individual microrafts. To isolate target cells, simply press the button on the controller which makes the release needle pierce through the lower surface of the QIAscout array and dislodge an individual microraft with your cell of interest attached. The released microraft is then recovered using a magnetic wand and transported to a secondary vessel (e.g., reaction tube of downstream application).

Steps in the QIAscout workflow include:

• Seeding and cultivating cells in medium on the QIAscout array
• Placing release device containing release needle on the microscope objective
• Placing array on the microscope stage
• Identifying microrafts containing single cells
• Piercing microraft containing cell of interest
• Transferring microraft to secondary vessel using a magnetic wand
• Processing the collected cell for downstream application or further cultivation

The cell distribution on the QIAscout array follows a Poisson distribution (see Table “Cell distribution on the array follows a Poisson distribution”). To achieve the maximum number of microrafts on the QIAscout array containing a single cell and to minimize the number of microrafts with no cell or more than one cell, it is suggested to plate cells at a cell : microraft ratio of 1:2 or 1:3. A QIAscout array contains 12,000 individual microrafts, and therefore plating 4,000–6,000 cells is generally recommended. Adjust the cell density of your suspension and add the desired number of cells to the QIAscout array in 2 ml of medium.

Cell distribution on the array follows a Poisson distribution

Cell : Well ratio	% Empty wells	% Wells capturing a single cell	% Wells capturing 2 or more cells
1:1	37	37	26
1:2	61	30	9
1:3	72	24	4
1:5	82	16	2
1:10	90	9	1

After adding cells to the QIAscout array, allow the cells to settle and wait until the cells have attached firmly enough to the microraft surface to allow for release and collection. A waiting time of 6–10 hours in culture is generally adequate, but the length of this period will be cell line-dependent. Once the cells are adhered to the microrafts, the QIAscout array can be screened under an inverted microscope to identify target cells and colonies and proceed with isolation and recovery.

The QIAscout is highly suitable for laboratories performing single-cell research. Single-cell isolation with QIAscout is compatible with multiple downstream applications such as:

• Whole Genome Amplification
• Whole Transcriptome Amplification
• Single-cell Sequencing
• Single-cell miRNA Analysis
• Clonal Expansion