Cultured normal cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), are prone to acquiring genetic abnormalities. Gene editing, such as CRISPR-Cas9 editing, may also generate cytogenetic changes. Cancer cells are particularly unstable. These changes may influence cell characteristics, potentially leading to inaccurate results in downstream applications. Cytogenetics provides an audit of genetic integrity.
What is array hybridization?
Array analysis utilizes slide-immobilized DNA probes which correspond to defined parts of the genome. DNA isolated from samples is fluorescently labelled and applied to these arrays, the resulting pattern of fluorescence is measured and compared to a physical control or a series of data sets in silico. A virtual map of probe copy number can be assembled based on the relative intensity of each probe. The results of this analysis are used for the detection and reporting of abnormalities that would be missed by traditional karyotyping methods.
Why perform array hybridization?
Array platforms offer a high-resolution analysis which allows for the detection and reporting of abnormalities that would be missed by G-banding. A much higher resolution of DNA dosage imbalances and loss of heterozygosity (LOH) can be characterized using array comparative genome hybridization (aCGH) and array genome hybridization (AGH). Although this type of analysis has higher definition than karyotype analysis, it completely misses important balanced translocations and has great limitations for detecting mosaic cell populations.
Nonetheless, G-banding and array analysis are complementary and should be used in combination to check that a cultured cell line remains chromosomally normal. Karyotype analysis is a low-resolution method of identifying large chromosomal abnormalities, including structural and numerical anomalies, as well as balanced translocations.
What array services do we offer?
Cell Guidance Systems offers two services for human samples based on different array platforms: aCGH (array comparative genomic hybridization) and AGH (array genomic hybridization).
aCGH is performed on the Agilent 8x60K platform: This system utilizes two differentially labeled DNA samples co-hybridized to an array with ~55,000 oligonucleotide probes. The relative abundance of DNA binding to each probe provides a direct measure of relative copy number of the two samples.
There are two service options for aCGH:
A single sample is hybridized against control DNA sample provided by ourselves.
Both test and control samples are provided by the customer and are co-hybridized to assess acquired copy number variations (CNVs). We recommend that both the control and test DNA samples are isolated from the cells at the same time.
AGH is performed using Affymetrix CytoScan 750K platform: The hybridization data from a single test DNA is compared with a series of control hybridizations in silico. The array has 750,486 distinct features. The AGH assay is particularly useful for assessing acquired loss of heterozygosity (LOH).
aCGH vs AGH array platforms
Average resolution (median probe spacing)
No Note 1
Copy number variation detection?
Single nucleotide polymorphism (SNP) probes
Loss of heterozygosity detection?
Mosaic detection sensitivity
Note 1 An in-silico comparison is performed against an extensive database of control samples
What types of abnormalities are reported?
Acquired copy number variations (CNVs) and loss of heterozygosity (LOH) present in at least ~15 – 20% of cells will be reported.
For the AGH analysis (Affymetrix CytoScan 750k platform), an assessment of normal variation is made with reference to ~5,000 normal control samples and a database of ~10,000 clinical samples. However, benign constitutional (heritable) CNVs will not be reported.
Balanced rearrangements and low level (10 – 20%) of mosaicism will not be detected using either array CGH or AGH. In practice, this level of mosaicism is statistically similar to the level which can be detected by karyotype analysis of 20 cells.
We recommend array analysis for identifying marker chromosomes and additional material on a chromosome of unknown origin. Karyotype analysis alone can detect certain acquired abnormalities, e.g. gain in the small arm of a chromosome, but the precise nature of certain unbalanced chromosomal abnormalities is difficult to establish by only using G-banding analysis.
Sample types accepted
The aCGH and AGH services are only available for human samples. Requirements for the samples are as listed below:
Sample type: isolated DNA
Concentration: >50 ng/µl
Minimum total volume: 1 µg
Purity: A260/A230 = 1.8 – 2.2 A260/A280 > 1.8
Service description and reporting
A summary report of the results will be issued following completion of the analysis. This will include the array profile overview, details of any abnormalities detected, and a description of the analysis carried out. This will be emailed to you in PDF format, along with a TIFF file containing an image of the genome overview.
The results are returned by email within 15 – 17 business days after the DNA samples have been received at our laboratories. Please note that lead times can be longer during busier periods. If this applies to your order, our team will keep you updated.
Frequently Asked Questions (FAQs)
For any additional questions, please refer to FAQs document here.