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Tips for Successful aCGH

Posted By Rosaria Esposito
Tags: CGH, Molecular Biology, Successful Research Tips

Enzo Life Sciences provides over 40 years of experience in the manufacturing and supply of research kits, biochemicals and biologicals. As Scientists Enabling Scientists, we realize the value in providing relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to share simple but useful hints for improving your daily tasks as well as the overall quality of your results. With this in mind, below is a list of tips for achieving high quality data by array Comparative Genomic Hybridization (aCGH), a powerful clinical diagnostic tool that can be used to evaluate copy number variation (CNV) changes in the genome. This list of suggestions is based not only on Enzo’s experience as a recognized pioneer in labeling and detection technologies, but also on solutions we offer on a regular basis, in order to assist researchers in obtaining the most accurate and consistent data for array CGH analyses.


aCGH: An Overview

The principle of aCGH is based on the quantitative comparison between a reference and a test DNA usually labeled with Cyanine 3 and Cyanine 5, green and red fluorescent dyes, respectively. Upon labeling, reference and test DNA are mixed and loaded onto a microarray chip, containing thousands of known target DNA sequences, for competitive hybridization. Based on the color of each spot in the array, CNVs throughout the genome can be detected.


Figure 1: Array CGH workflow.


Definitions

Copy Number Variation (CNV):
Gene gain or loss associated with duplications or deletions of a chromosomal region. These chromosomal abnormalities often cause genetic diseases, neurological disorders, as well as cancers.

Single-Nucleotide Polymorphism (SNP):
Variation of a single nucleotide occurring in a specific position in the genome.

Dye incorporation:
pmol of dye incorporated per reaction.

Specific activity:
pmol of dye incorporated per µg of DNA.

Signal intensity:
Measurement of overall fluorescence intensity. The value should be above 200.

Background noise:
Standard deviation of the background-subtracted signals of the negative control probes. The value should be below 25.

Signal-to-noise:
ratio between the probe signal and the background noise. The value should be above 30.

Derivative log ratio (DLR):
A measure of array quality indicative of the variation of the signal around the mean. The value should be below 0.2.



1. Storage and handling

Familiarizing yourself with the manual is the first important step towards success. Before starting, make sure that the protocol is understood, the critical steps identified, and the availability of the required tools verified. Look for the correct storage instructions for all the materials you are going to use. For example, labeling kits components should be conveniently aliquoted to avoid freeze/thaw cycles, and kept at -20°C. The Cyanine 3- and Cyanine 5-labeled deoxynucleotide mixes are light sensitive and therefore need to be protected from light exposure at all times. If you are using a column-based DNA purification kit, you may need to store it at room temperature. Proper storage and handling are fundamental to maximizing the performance of the products and minimizing the risk of failures.


2. Start with good quality DNA: check for purity

DNA labeling is one of the most critical steps of the aCGH workflow, since the quality of the results are strictly dependent on the quality of the probes hybridized on the microarray. High quality and high molecular weight genomic DNA should therefore always be used, since it will directly affect the efficiency of the labeling reaction. Once the genomic DNA has been isolated with the method of your choice, concentration and purity will have to be evaluated. The two most important parameters to take into account will be the ratio of the absorbance at 260 vs 280nm, and at 260 vs 230nm. The A260/280 ratio, used to assess protein contamination, should be >1.8. The A260/230 is related to the possible presence of contaminants absorbing at 230nm, such as organic compounds (i.e. the phenol used for DNA extraction) or chaotropic salts (e.g. guanidinium chloride). This value should be in the range of 2.0–2.2. In case of significant deviation from these ratios, you should consider re-purifying your sample. Alternatively, you could try and use a higher DNA input for the labeling reaction.


3. Start with good quality DNA: check for integrity

You can use a classical gel electrophoresis or automated electrophoresis systems to verify the integrity of the genomic DNA. In order to avoid freeze/thaw cycles, which can easily break the chromosomes, you should store it at 4°C. An alternative is to freeze aliquots at -20°C, defrost when needed, and keep the aliquot in use at 4°C for short term storage.


4. Choose the right labeling kit

Will you be looking for SNPs? Are you handling low input samples? Do you need a high genome coverage? Or would you rather run more tests at once? When setting up your aCGH, think about these questions to choose the appropriate labeling kit, the right microarray format and thus, the adapted protocol. Enzo’s proprietary labeling technology allowed the development of different labeling kits for aCGH to accommodate the different experimental needs of the end user. The CYTAG® TotalCGH Labeling Kit allows single nucleotide polymorphism (SNP) arrays in addition to standard CGH arrays. The CYTAG® SuperCGH Labeling Kit is specifically designed for samples with limited amounts of starting material (e.g. 50 ng of DNA input).


5. Choose the correct array type

Mosaicism Detection

Genomic aberrations smaller than 10Mb can be successfully detected in samples with as low as 10% mosaicism with Enzo’s CYTAG® SuperCGH Labeling Kit. Please read the Application Note for more information.
Different array formats are currently available, depending on the resolution allowed by the target probes present on the chip. The most common are the 8x60K and 4x180K CGH arrays. In the first case, the slide is divided into eight areas, each one containing 60,000 probes, making it possible to test eight samples in the same experiment. The second type of slide is divided into four areas, containing 180,000 probes. The resolution for this type is higher with a greater genome coverage, but only four samples can be run on the same chip.


6. Set the labeling reaction properly

Dye Swap

The “dye swap” consists of switching the dye used to label reference and test DNA. This is very useful to confirm the presence of uncharacterized anomalies and aberrations, thus excluding the possibility of a dye bias.
Carefully follow manufacturer guidelines, respecting volumes, concentrations, timing, and incubation temperatures. The labeling reaction of Enzo’s kits are based on the incorporation of cyanine dyes into newly synthesized DNA by random priming. Both the denaturation and the primer extension steps are absolutely crucial in this process. Changing, and in particular, shortening the reaction time will cause an inefficient and incomplete incorporation of Cyanine-labeled deoxynucleotides.


7. Choose the purification method

Probe Purification by Alcohol Precipitation

DNA is soluble in water. A solution of salts (NaOAc or NaCl) and alcohol (ethanol or isopropanol) will make it precipitate. When using this technique to purify probes, ethanol should be preferred over isopropanol, to better remove free nucleotides. NaOAc will work better than NaCl to get a cleaner labeled DNA.
The labeled DNA will need to be purified in order to remove unincorporated nucleotides, buffers, etc. One of the most common ways is to use silica membrane-based purification columns (for example the PCR & Gel Clean-up Kit, included in Enzo’s CYTAG® TotalCGH and SuperCGH labeling kits) or alternatively, columns containing cellulose membranes to filter labeled samples. In some laboratories, the purification is obtained by a classical DNA precipitation (e.g. ethanol and sodium acetate solution). Although DNA yield is related to the type of purification, Enzo’s CYTAG® labeling kits are compatible with each one of these methods. The choice will mainly depend on the experimental needs of the end user (timing, protocols, instruments availability, etc.).


8. Check the quality of your probes

Before proceeding with the hybridization on the array, it is worth checking the efficiency of the labeling reaction in order to avoid wasting a relatively expensive chip with poor quality material. This is typically done with a NanoDrop in Microarray Measurement Mode and dsDNA settings to quantify the DNA yield, the dye incorporation and the specific activity. High dye incorporation correlates with increased accuracy of variant detection and minimized manual data analysis. Enzo’s CYTAG® SuperCGH Labeling Kit has been optimized to get the best performance with as low as 50 ng of input DNA. When using the labeling protocol for 4x180k arrays, the DNA yield should be greater than 5.0 µg of DNA. The DNA that has been generated should contain at least 300 pmoles of incorporated Cyanine 3 or at least 200 pmoles of incorporated Cyanine 5. The specific activity should therefore be at least ~60 pmol/µg and ~40 pmol/µg for Cyanine 3 and Cyanine 5, respectively.


Figure 2: 50 ng DNA samples were labeled with Enzo's CYTAG® SuperCGH Labeling Kit for Oligo Arrays and a leading competitor's kit. Enzo's proprietary labeling technology generates superior dye incorporation and the highest specific activity of labeling for Cy3 and Cy5.
Ozone effects on Cy5

Elevated environmental temperatures are associated with an increase in ozone levels. This phenomenon can affect Cy5, but not Cy3, signal intensity.



9. Hybridization procedure

Wave Effect

A microarray technical artifact is visualized as a wavy pattern of the hybridization intensities along the chromosome. This can be due to GC content of the probe and the potential bias during DNA isolation. Important parameters to take into account in order to limit this effect is the amount of Cot-1 DNA and the denaturing step prior to labeling.
Prepare the hybridization mix strictly following the instructions of the array manufacturer. Based upon the requirements of your hybridization platform, volume reduction may be necessary (e.g. you need half of the volume for the 8x60K array with respect to the 4x180k format). For your convenience, Enzo’s CYTAG® labeling kit manuals contain specific protocols, with adjusted reaction volumes for DNA labeling when using 8x60K or 4x180k arrays. Alternatively, it is also possible to concentrate the samples via centrifugal vacuum or by DNA precipitation. When preparing the hybridization mix, pay attention to the amount of Cot DNA (DNA fragments enriched in repetitive sequence), which is the key element in blocking non-specific interactions. The buffer stringency and the temperature of incubation will also affect the quality of the final results. Incorrect hybridization conditions can lead to low signal, elevated background and poor signal-to-noise ratio.


10. Interpret the results

The microarray scanner will produce an image file that can be analyzed with different dedicated programs. The software generates a report displaying QC metrics, essential to guarantee the reproducibility and the reliability of the assay. Parameters such as the background noise, the signal-to-noise ratio, the signal intensity and the Derivative Log Ratio (DLR) are evaluated and a score is automatically attributed. They are all related to the efficiency of the labeling and the hybridization. In particular, the DLR score is considered to be the key QC parameter as it is an indication of the noise around the mean signal of the array. DLR scores lower than 0.2 allow a more accurate identification of multiplied or deleted chromosome regions, thereby reducing the need for experimental repeats. High DLR values can be directly correlated with poor DNA quality and/or labeling efficiency. Enzo’s proprietary labeling technology provides superior DLR scores (0.09-0.12), even with low DNA input, typically associated with difficult (and precious) samples, such us biopsies or amniotic fluid.


Figure 3: : CYTAG® SuperCGH Labeling Kit labels DNA across a dynamic analytical range. From 4 different amniotic prenatal samples, 50 ng DNA input were labeled and applied to an 8 x 60K array, showing consistently low DLR scores.

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