Microarray-CGH for the Assessment of Aneuploidy in Human Polar Bodies and Oocytes

Part of the Methods in Molecular Biology book series (MIMB, volume 957)

Abstract

The cytogenetic analysis of single cells, such as oocytes and polar bodies, is extremely challenging. The main problem is low probability of obtaining a metaphase preparation in which all of the chromosomes are sufficiently well spread to permit accurate analysis (no overlapping chromosomes, no chromosomes lost). As a result, a high proportion of the oocytes subjected to cytogenetic analysis are not suitable for traditional chromosome banding studies or for molecular cytogenetic methods such as spectral karyotyping (SKY) or multiplex fluorescence in situ hybridization (M-FISH). Fortunately, recent innovations in whole genome amplification and microarray technologies have provided a means to analyze the copy number of every chromosome in single cells with high accuracy. Here we describe the use of such methods for the investigation of chromosome and chromatid abnormalities in human oocytes and polar bodies.

Key words

Whole genome amplification Microarray Comparative genomic hybridization Aneuploidy Polar body Oocyte 

1 Introduction

The cytogenetic assessment of human oocytes has attracted a great deal of attention from scientists and clinicians alike. Not only is the meiotic process of fundamental biological interest, but the outcome of abnormal chromosome segregation has profound medical consequences. Aneuploidy, mostly oocyte derived, is the leading cause of first trimester miscarriage, congenital abnormalities, and mental retardation (e.g., Down syndrome). It is also believed to be the underlying reason for many unsuccessful infertility treatments.

Loss or gain of chromosomes or chromatids during the first and/or second meiotic divisions (i.e., meiosis I and meiosis II) is extremely common in our species. Indeed, the frequency at which errors of this type occur is an order of magnitude higher in human oocytes compared with most other mammalian species. Malsegregation of chromosomes and chromatids is associated with female age, increasing slowly during early adulthood, but accelerating from the mid-30s onwards. Typically, about 10% of oocytes from women under 30 are found to be abnormal, while more than two-thirds derived from women in their 40s are aneuploid (1, 2, 3, 4). Most abnormalities involve premature separation of sister chromatids (these should normally remain attached to each other until anaphase of meiosis II). Failure to maintain sister chromatid cohesion leads to separation during the first meiotic division, followed by segregation to either spindle pole at random (2, 4, 5). In young women most errors occur during meiosis I, but for women towards the end of their reproductive life the second meiotic division may be a more important contributor to abnormality (4).

A great deal of research into oocyte chromosomes has been undertaken using conventional cytogenetic techniques such as G-banding and R-banding (1). However, such approaches are limited by the difficulty in obtaining a good quality chromosome spread from a single oocyte. Many of the oocytes assessed cannot be analyzed due to overlapping chromosomes, whereas other spreads are found to be missing large numbers of chromosomes. It can be difficult to be sure if a single chromosome loss is a consequence of a true meiotic error or an artifact of the spreading procedure.

An alternative to conventional chromosome banding approaches is to use fluorescent in situ hybridization (FISH). This method has the advantage that each chromosome is identified by a fluorescent probe with a distinct color, so chromosomes that are overlapping or of poor morphology can still be readily identified (2). However, the main drawback of the FISH approach is that only a handful of chromosomes can be assessed in each oocyte, due to the limited range of spectrally distinct fluorochromes (i.e., few colors) available for probe labeling. Although it is possible to use molecular cytogenetic methods related to FISH, such as Multiplex-FISH (M-FISH) or spectral karyotyping (SKY), to identify each of the oocyte chromosomes, in practice this runs into the same problems as chromosome banding approaches, since good quality metaphase spreads are needed (6).

In terms of a simple assessment of chromosome/chromatid losses and gains, microarray comparative genomic hybridization (microarray-CGH) is the best method currently available for oocyte studies. Microarray-CGH is a DNA-based technique, so there is no need to obtain chromosome spreads from the sample; rather the cell is placed inside a microcentrifuge tube. The oocyte DNA is amplified using a whole genome amplification technique; the DNA is then labeled and applied to a microarray comprising numerous probes for specific chromosomal regions affixed to a solid support (usually a glass slide); a “reference” DNA sample derived from a chromosomally normal individual, labeled in a different color is applied to the microarray at the same time. Detection of loss or gain of chromosomal material involves computer assisted analysis of the color of each probe on the microarray. An equal quantity of fluorescence corresponding to the oocyte DNA and the reference DNA is indicative of normality for the chromosomal region corresponding to the probe; a chromatid/chromosome gain is revealed by an excess of fluorescence attributable to the oocyte; a deficiency of fluorescence from the oocyte relative to the reference DNA demonstrates a loss of chromosomal material in the oocyte. This approach has the potential to provide a highly accurate analysis of every chromosome. However, most commercially available microarray platforms are not suitable for single cell analysis, so it is advised to only use those that have been validated at the single cell level.

This chapter describes the use of the 24Sure microarray manufactured by BlueGnome (Cambridge, UK). This microarray has been highly optimized and validated for use with single cells, including oocytes and polar bodies (PBs) (4, 7, 8, 9). We routinely use this microarray for analyzing polar bodies biopsied from human oocytes as part of our preimplantation genetic screening (PGS) program. Given that the oocyte and PB are “daughter cells” of meiosis I, any abnormality detected in a PB is expected to be reciprocated in the corresponding oocyte (e.g., a loss of a chromosome in the first polar body is associated with a gain of the same chromosome in the oocyte after completion of meiosis I). The aim of PGS is to identify chromosomally normal oocytes, produced for the purpose of in vitro fertilization (IVF) treatment. Embryos derived from the chromosomally normal oocytes are then prioritized for transfer to the uterus with the hope of increasing pregnancy rates and decreasing the risks of miscarriage and Down syndrome. Embryos derived from abnormal oocytes are not transferred.

The 24Sure microarray is produced by “spotting” of more than 3,200 bacterial artificial chromosomes (BACs) onto a glass slide. The BACs have been carefully selected on the basis of having little variation in over 5,000 hybridizations, an absence of known copy-number variations (CNVs) and the highest levels of reproducibility and sensitivity in conjunction with whole genome amplification. Purpose-designed software (BlueFuse Multi) smooth results, allowing robust detection of chromosome losses and gains. If analysis of smaller chromosomal regions is desired, such as chromosome fragments associated with reciprocal translocations, it is recommended to use the higher density 24Sure+ microarray (7, 9). The 24Sure+ microarray has an average resolution of 2–5 Mb compared to 10 Mb for standard 24Sure microarrays. In particular, 24Sure+ has greater coverage of sub-telomeric and peri-centromeric regions, enabling accurate characterization of arm level aneuploidy and other large-scale structural abnormalities.

2 Materials

The SurePlex protocol should be carried out in a sterile (DNA-free, UV-irradiated) laminar flow cabinet in a separate room to where amplified products are handled, in order to prevent sample contamination. All amplification and labeling reactions should be set up on ice (unless indicated otherwise). Prepare washing and hybridization solutions using ultrapure water (18 MΩ cm at 25°C) and molecular biology grade reagents. Prepare and store all solutions at room temperature unless indicated otherwise by the manufacturer. Follow all waste disposal regulations carefully when disposing of materials.

2.1 Cell Preparation, Lysis, and Whole Genome Amplification

  1. 1.

    Collection of oocytes or PBs: Dulbecco’s phosphate buffered saline (DPBS) with 0.1% polyvinyl alcohol (PVA) (Sigma-Aldrich).

     
  2. 2.

    SurePlex DNA Amplification System (Rubicon Genomics Inc., Ann Arbor, Michigan, USA), including: Cell Extraction Buffer, Extraction Enzyme Dilution Buffer, Cell Extraction Enzyme, Pre-amp Buffer, Pre-amp Enzyme, Amplification Buffer, Amplification Enzyme, and Nuclease-free water.

     
  3. 3.

    1× Tris–Borate–EDTA (TBE) Buffer: 89 mM Tris base, 89 mM Boric acid, 2 mM EDTA, pH 8.3  ±  0.1.

     
  4. 4.

    One percent agarose gel: Add 1 g agarose to 100 ml 1× TBE buffer and heat in microwave until dissolved. Allow to cool to room temperature then add 2 μl of Ethidium Bromide (10 mg/ml).

     
  5. 5.

    10× Blue Juice gel loading buffer (Invitrogen, CA, USA).

     
  6. 6.

    100 base pairs (bp) Lower Scale DNA Ladder (Fisher Scientific Ltd, UK).

     
  7. 7.

    Benchtop microcentrifuge (appropriate size for 0.2 ml tubes).

     
  8. 8.

    Thermal cycler.

     
  9. 9.

    UV transilluminator/gel imaging system.

     

2.2 Labeling and Hybridization

  1. 1.

    Fluorescent Labeling System (BlueGnome Ltd, Cambridge, UK), including: dCTP-labeling reagents, SureRef Reference Male DNA, and COT Human DNA.

     
  2. 2.

    24Sure microarrays or 24Sure+ microarrays (BlueGnome Ltd, Cambridge, UK).

     
  3. 3.

    Formamide.

     
  4. 4.

    Centrifugal evaporator.

     
  5. 5.

    20× Saline-Sodium Citrate (SSC) buffer and Tween 20.

     
  6. 6.

    2× SSC/50% formamide: Prepare 6 ml by mixing 3 ml of formamide with 600 μl of 20× SSC and 2.4 ml of water.

     
  7. 7.

    Humidified box to be used as a hybridization chamber: Use a box that can be closed off completely such as a slide box or a sandwich box. Line the inside with tissue saturated with 6 ml of 2× SSC/50% formamide.

     
  8. 8.

    Parafilm.

     
  9. 9.

    Lidded non-circulating water bath at 47°C.

     
  10. 10.

    Square coverslips.

     
  11. 11.

    Wash 1 (2× SSC/0.05% Tween20): Prepare 1,000 ml by adding 100 ml 20× SSC (pH 7.0) and 0.5 ml Tween 20 to 899.5 ml ultrapure water. Prepare all washing solutions fresh on the day of use.

     
  12. 12.

    Wash 2 (1× SSC): Prepare 500 ml by adding 25 ml 20× SSC (pH 7.0) to 475 ml ultrapure water.

     
  13. 13.

    Wash 3 (0.1× SSC): Prepare 1,000 ml by adding 5 ml 20× SSC (pH 7.0) to 995 ml ultrapure water.

     
  14. 14.

    Coplin jars.

     
  15. 15.

    Stainless steel slide rack.

     
  16. 16.

    Square glass staining dish.

     
  17. 17.

    Magnetic stirrer.

     
  18. 18.

    2.5 cm magnetic stir bar.

     
  19. 19.

    Water bath with good temperature stability. We recommend the Hybex Microarray Incubation System with water bath insert (BlueGnome 4303-1).

     

2.3 Microarray Scanning and Analysis

  1. 1.

    A two-channel microarray scanner (e.g., InnoScan 700, Innopsys, France) for capturing the Cy3 and Cy5 signals produced by the independently labeled test and control DNA samples.

     
  2. 2.

    MAPIX software for scanning the microarray slides.

     
  3. 3.

    BlueFuse Multi data analysis software (BlueGnome Ltd, Cambridge, UK) for analyzing scanned images.

     

3 Methods

The 24Sure protocol described in this chapter involves whole genome amplification using the SurePlex DNA Amplification System and takes a minimum of 8 h and 40 min to complete; however, certain steps (indicated with *) can be extended to allow the procedure to fit into a standard working day. The approximate timings for each step are shown in Table 1. The protocol used for the high-resolution 24Sure+ microarrays is essentially identical.
Table 1

Summary of steps involved in the microarray-CGH protocol

Protocol step

Approximate time

Subheadings

Sample preparation

30 min

3.1

Amplification

2 h

3.1

Labeling*

1.5–4 h (can be left overnight)

3.2

Combination and volume reduction

1 h

3.3

Hybridization*

3–16 h (can be left overnight)

3.4

Post-hybridization washes

30 min

3.5

Scanning and analysis

10 min (for a single slide, 2 samples)

3.6

3.1 Sample Preparation

  1. 1.

    Collect the oocytes or PBs in DNase-free, thin-walled 0.2 ml PCR tubes in 1–2 μl of PBS (0.1% PVA) (see Notes 1 and 2).

     
  2. 2.

    Wash oocytes through at least three 5 μl droplets of PBS pipetted onto a clean petri dish before transfer to the PCR tube. Washing is recommended in order to remove any DNA contaminants that may have been present in the medium in which the sample had been cultured.

     
  3. 3.

    If not used immediately, samples should be stored at −80°C and processed within 6 months. Once thawed, keep the oocytes or PBs at 4°C or on ice at all times. Avoid repeated cycles of freezing and thawing (see Note 3).

     
  4. 4.

    Prepare the oocytes or PBs for lysis by centrifuging the tube(s) at 200  ×  g for 3 min or pulse centrifuge briefly.

     
  5. 5.

    Add 3 μl of Cell Extraction Buffer to each sample including any possible controls (see Note 4). The total volume in the tube should be 4–5 μl.

     
  6. 6.

    Prepare the extraction mastermix by adding 0.2 μl Cell Extraction Enzyme to 4.8 μl Extraction Enzyme Dilution Buffer per sample. Mix by flicking the mastermix tube and centrifuging briefly to collect the fluid at the bottom of the tube. The mastermix is prepared for an extra 10% of tubes, e.g., if analysis of two samples is to take place, the mastermix is prepared for 2.2 tubes.

     
  7. 7.

    Add 5 μl of freshly prepared extraction mastermix to each of the 0.2 ml PCR tubes containing the 4–5 μl sample.

     
  8. 8.

    “Pulse” centrifuge the tubes for approximately 10 s to ensure collection of the extraction mix and sample at the bottom of the tube.

     
  9. 9.
    Incubate the tubes in a thermal cycler using the program shown in Table 2 (see Notes 5 and 6).
    Table 2

    Thermal cycler program for the SurePlex lysis step

    Number of cycles

    Temperature (°C)

    Time (min)

    1 Cycle

    75

    10

    1 Cycle

    95

    4

    1 Cycle

    Room temperature

    Hold

     
  10. 10.

    Place the samples on ice while setting up and adding the pre-amplification mastermix.

     

3.2 Pre-amplification

  1. 1.

    Prepare the Pre-Amp mastermix by adding 0.2 μl SurePlex pre-amp enzyme to 4.8 μl SurePlex pre-amp buffer per sample and mixing well. As with the lysis step, the mastermix is prepared for an extra 10% of tubes to allow for loss of fluid during pipetting.

     
  2. 2.

    Add 5 μl of SurePlex Pre-Amp mix to 10 μl of sample. Briefly centrifuge the tubes to ensure that all contents have collected at the bottom of the tube.

     
  3. 3.
    Incubate the tubes in a thermal cycler using the program shown in Table 3 (see Note 2).
    Table 3

    Thermal cycler program for the SurePlex pre-amplification step

    Number of cycles

    Temperature (°C)

    Time

    1 Cycle

    95

    2 min

    12 Cycles

    95

    15 s

     

    15

    50 s

     

    25

    40 s

     

    35

    30 s

     

    65

    40 s

     

    75

    40 s

    1 Cycle

    4

    Hold

     
  4. 4.

    Place the pre-amplified products on ice.

     

3.3 Amplification

  1. 1.

    Prepare the amplification mastermix by adding 0.8 μl SurePlex amplification enzyme to 25 μl SurePlex amplification buffer and 34.2 μl nuclease-free water per sample and mixing well by vortexing for 5 s. The mastermix is prepared for an extra 10% of tubes, e.g., if analysis of two samples is to take place, then the mastermix is prepared for 2.2 tubes.

     
  2. 2.

    Add 60 μl of SurePlex Amplification mix to the 15 μl of synthesis reaction product and mix well by flicking and briefly pulse centrifuging for 10 s.

     
  3. 3.
    Incubate the tubes in a thermal cycler using the program shown in Table 4 (see Note 7).
    Table 4

    Thermal cycler program for the SurePlex amplification step

    Number of cycles

    Temperature (°C)

    Time

    1 Cycle

    95

    2 min

    14 Cycles

    95

    15 s

     

    65

    1 min

     

    75

    1 min

    1 Cycle

    4

    Hold

     

3.4 Checking Amplification Success Using Agarose Electrophoresis

If whole genome amplification has not been successful, microarray analysis will not yield usable data (see Note 8 and Fig. 1). As microarrays are relatively expensive, it is desirable to avoid wasting them on samples that have not amplified.
Fig. 1.

SurePlex amplified DNA detected on a 1% agarose 1× TBE gel. The 100 bp Lower Scale DNA Ladder (L) was loaded in the first lane followed by the amplified polar bodies. (a) The smears observed for the four polar bodies between 200 and 2,000 bp indicate successful whole genome amplification. (b) The smears observed for samples 1, 2, 3, 5, 7, 8, 9, 10, and 11 demonstrate successful amplification. The weaker smear observed for polar body 6 indicates poor amplification. This sample is likely to give noisy microarray results that may be difficult to interpret. No amplification was detected for the PB in lane 4.

  1. 1.

    Mix 5 μl of each amplified sample with 0.5 μl gel loading buffer.

     
  2. 2.

    Load the samples on a 1% agarose gel alongside 5 μl of 100 bp Lower Scale DNA Ladder (or equivalent).

     
  3. 3.

    Electrophorese at 175–190 V for 10 min.

     
  4. 4.

    Visualize the products using a UV transilluminator/gel imaging system. Successful amplification should result in a smear that ranges between 100 and 2,000 base pairs (bp) with strongest intensity around 500 bp (see Fig. 1).

     

3.5 Labeling and Preparation of Amplified Test and Reference DNA Samples

The amplified test and reference DNAs are labeled with Cy3 (green) and Cy5 (red) fluorophores, respectively, using random primers. The SureRef, normal male (46,XY) reference DNA, is well matched to amplified single cells or PBs and is used as a hybridization control (see Note 9).
  1. 1.

    Thaw, vortex, and centrifuge briefly the components of the Fluorescent Labeling System and keep on ice. Prepare the labeling mixes by adding the following components in the order they are listed.

     
  2. 2.

    Prepare mastermix for the test samples (Cy3 labeling mix) by adding the following components in the order they are listed: 5 μl Reaction Buffer, 5 μl Primer Solution, 5 μl dCTP-Labeling Mix, and 1 μl Cy3 dCTP. The mastermix is prepared for an extra 10% of tubes.

     
  3. 3.

    Prepare mastermix for the reference samples (Cy5 labeling mix) by adding the following components in the order they are listed: 5 μl Reaction Buffer, 5 μl Primer Solution, 5 μl dCTP-Labeling Mix, 1 μl Cy3 dCTP, and 8 μl SureRef DNA.

     
  4. 4.

    Aliquot 16 μl of the Cy3 labeling mix into clearly labeled PCR tubes. One tube is required per test sample.

     
  5. 5.

    Add 8 μl of the amplified DNA sample into the appropriate tube and mix well by pipetting up and down.

     
  6. 6.

    Aliquot 24 μl of Cy5 labeling mix into an equal number of “reference” PCR tubes.

     
  7. 7.

    Place all of the tubes in a thermal cycler (with a preheated lid) at 94°C for 5 min to denature the DNA.

     
  8. 8.

    Immediately transfer tubes onto ice or a pre-cooled thermal cycler at 4°C for another 5 min.

     
  9. 9.

    Add 1 μl of Klenow enzyme to each test and reference sample.

     
  10. 10.

    Mix well by flicking and then pulse centrifuge for 5 s to collect all of the fluid in the bottom of the tubes. Place the tubes in a thermal cycler (with a preheated lid) for 2–4 h at 37°C (see Note 10). Incubation is generally performed for 3 h, but labeling can be completed in as little as 1.5 h, or left overnight if more convenient.

     
  11. 11.

    Once the incubation is complete, place all tubes on ice.

     

3.6 Combination of the Fluorescently Labeled Products and Volume Reduction

In these steps, the labeled test and reference DNAs are combined and the volume reduced using a centrifugal evaporator. Alternatively, ethanol precipitation can be used to reduce the volume (see Note 11). The labeled DNAs will be hybridized onto the probes affixed to the microarray slides.
  1. 1.

    Preheat the centrifugal evaporator to 75°C for 30 min.

     
  2. 2.

    Combine the labeled test and reference DNA samples by transferring the 25 μl of Cy5-labeled reference DNA to the 0.2 ml PCR tube containing the Cy3-labeled test sample.

     
  3. 3.

    Add 25 μl COT Human DNA to each tube containing the combined Cy3/Cy5 products.

     
  4. 4.

    Centrifuge the tubes (the centrifugal evaporator only has a single speed) at 75°C, with the lids open, for approximately 40 min or until most of the liquid volume is evaporated and only roughly 3 μl remain in each tube (see Note 12).

     

3.7 Probe Preparation, Denaturation, and Hybridization

  1. 1.

    Resuspend each pellet in 21 μl of preheated DS hybridization buffer.

     
  2. 2.

    Mix by flicking the tube and/or vortexing, then pulse centrifuge for approximately 5 s to collect the fluid in the bottom of the tube.

     
  3. 3.

    Denature the probes in a thermal cycler at 75°C for 10 min, pulse centrifuge again for approximately 5 s, and allow to cool to room temperature.

     
  4. 4.

    Assign a sample to each hybridization area. The 24Sure technology supports the hybridization of two samples on each slide.

     
  5. 5.

    Load 18 μl of each probe onto a square coverslip for each hybridization area (a paper hybridization template is provided with the microarrays to help with the positioning of the coverslips).

     
  6. 6.

    Lower the slide facing downwards (barcode at the bottom) until it comes into contact with the coverslip.

     
  7. 7.

    After the probe mixture spreads out between the slide and the coverslip it is possible to lift the slide and turn it face-up.

     
  8. 8.

    Place the 24Sure slides in the prepared hybridization chamber, close the lid firmly, seal it with parafilm, and float the chamber in a non-circulating lidded water bath for 3–16 h at 47°C. This incubation is generally carried out overnight.

     

3.8 Post-hybridization Washes

24Sure slides are washed to remove any un-hybridized DNA. A high temperature, formamide-free wash is used to deliver the correct levels of stringency.
  1. 1.

    Briefly wash the 24Sure slides, agitating them manually in a coplin jar containing Wash 1 solution at room temperature. Ideally, the coverslips will float off during this wash, but in some cases they will need to be gently removed by hand.

     
  2. 2.

    After removal of coverslips, immediately transfer each slide to a stainless steel slide rack placed inside a square glass staining dish containing 400 ml of Wash 1 and a 2.5 cm magnetic stir bar (room temperature).

     
  3. 3.

    Once the rack is fully loaded, cover the dish and wash the slides for 10 min using a magnetic stirrer.

     
  4. 4.

    Transfer the slides in the rack to another square glass staining dish containing 400 ml of Wash 2 and wash for another 10 min on the magnetic stirrer (room temperature).

     
  5. 5.

    Transfer the slides to the Hybex Microarray Incubation System containing 400 ml of Wash 3 preheated for 30 min to 59°C and incubate for 5 min.

     
  6. 6.

    Finally, transfer the slides to a square glass staining dish containing 400 ml of Wash 3 and wash for 1 min using the magnetic stirrer.

     
  7. 7.

    Dry the slides by centrifuging at 170  ×  g for 3 min and then store in a slide box at room temperature (see Note 13).

     

3.9 Microarray Scanning and Analysis

  1. 1.

    Connect the MAPIX software to the scanner.

     
  2. 2.

    Place the slide in the scanner with the barcode (and hybridization area) facing upwards.

     
  3. 3.

    Select “Preview” under the Tools menu to pre-scan and obtain a low resolution image of the entire slide allowing the identification of the hybridization areas and detection of the barcode.

     
  4. 4.

    Once the pre-scan is complete, select a hybridization area and scan it at high resolution (see Note 14).

     
  5. 5.

    Save the resulting TIFF image and name the file including the barcode and sample number/sample name.

     
  6. 6.

    Using the BlueFuse Multi data analysis software, open the “Sample Editor” and add the details of each sample (e.g., date and method of extraction) to analyze. It is recommended to enter sample details into the BlueFuse Multi database in order to facilitate information retrieval and future analysis.

     
  7. 7.

    Click the “Run Batch Processing” button to complete the analysis of all unprocessed samples. The obtained results can be viewed and interpreted (see Note 15).

     

3.10 Interpretation Criteria

The following is a list of the important criteria used for determining chromosomal constitution:
  1. 1.

    The data obtained from the fluorescence ratios of the reference DNA and the test DNA are presented as a deviation from the Log2 ratio between Cy3 and Cy5 signals for each target in the BAC microarray.

     
  2. 2.

    The BlueFuse Multi software establishes the normal ranges at Log2 ratio of +0.38 and −0.38.

     
  3. 3.
    Values for normal chromosome copy number should fall between +0.38 and −0.38 for the DNA sequences of the whole chromosome (Fig. 2).
    Fig. 2.

    Microarray-CGH analysis of a normal (23,X) polar body. The apparent gain of X-chromosome material and loss of Y-chromosome material is seen because a male reference DNA was used (SureRef, BlueGnome). If a female reference DNA had been used no losses or gains would have been observed.

     
  4. 4.

    A Log2 ratio greater than +0.38 denotes gain of DNA and a Log2 ratio below −0.38 denotes loss of DNA.

     
  5. 5.
    In most cases, a profile shift resulting in Log2 ratios above +0.4 for the whole chromosome indicates a gain of that chromosome (see Note 16); whereas a shift resulting in Log2 ratios between +0.25 and +0.4 indicates the gain of a single chromatid (Figs. 3 and 4).
    Fig. 3.

    Microarray-CGH analysis of highly abnormal first (a) and second (b) polar bodies sequentially biopsied from the same oocyte. (a) The first polar body has lost one chromatid belonging to chromosomes 4, 7, and 17 and has one extra chromatid for chromosomes 14, 15, 20, 21, and 22. The polar body has also lost the entire chromosome 16 (both chromatids). (b) The second polar body has gains of chromatids 1, 2, 4, 13, and 16 and losses of chromatids 6, 11, 14, and 19. The corresponding oocyte was therefore aneuploid (22,X,−1,−2,+6,+7,+11,−13,−15,+16,+17,+19,−20,−21,−22).

    Fig. 4.

    Microarray-CGH analysis of aneuploid first (a) and second (b) polar bodies sequentially biopsied from the same oocyte. (a) The first polar body has one extra chromatid for chromosome16. (b) The second polar body has lost chromatid 16. The corresponding oocyte was therefore euploid (23, X).

     
  6. 6.

    A profile shift resulting in Log2 ratios of −0.9 or below indicates loss of the entire chromosome (see Note 17); whereas a shift between −0.40 and −0.80 indicates the loss of a single chromatid (Fig. 3).

     

4 Notes

  1. 1.

    If analyzing oocytes the zona pellucida should be removed prior to whole genome amplification. This is because somatic cells (cumulus cells) and surplus spermatozoa are often stuck to the outer surface of the zona pellucida after IVF and their DNA is likely to be amplified along with the oocyte DNA. This contamination will prevent accurate analysis of the oocyte. The zona pellucida can be removed by brief immersion in pronase or acidified Tyrode solution.

     
  2. 2.

    The volume of PBS should not exceed 2 μl or the lysis and amplification reagents will be diluted to an unacceptable level. A volume of 1–1.5 μl is ideal.

     
  3. 3.

    For storage of samples for more than 6 months it is recommended that the cell is lysed and the DNA amplified. The resulting SurePlex products can be frozen and stored for extended periods (>12 months).

     
  4. 4.

    It is critical to use good laboratory practice and minimize pipetting errors throughout the protocol. This is particularly important during the amplification step, as any contaminants may also be amplified and could affect the results.

     
  5. 5.

    Make sure that the pipette tip does not touch the bottom of the tube when adding the lysis buffer or the extraction mastermix.

     
  6. 6.

    Most “whole genome amplification” techniques do not truly amplify the entire genome. Some sequences may be absent or under-represented, while others may be relatively over-amplified. Consequently it is important to use a microarray that has been optimized for use with the chosen WGA method and consists of probes situated in regions of the genome that are represented after amplification. The 24Sure microarray has been designed to be compatible with SurePlex DNA amplification.

     
  7. 7.

    Always use a heated lid on the thermal cycler. Do not carry out reactions under oil.

     
  8. 8.

    Typically, 5% of biopsied cells fail to amplify. The most likely causes of amplification failure are the presence of degraded DNA in the biopsied PB and failure to transfer.

     
  9. 9.

    It is essential that the reference DNA is prepared in the same way as the sample DNA. It is especially important for both DNAs to be amplified using the same method (i.e., SurePlex). The SureRef DNA, supplied by the manufacturer, is derived from a chromosomally normal male and has been subjected to SurePlex amplification.

     
  10. 10.

    Half an hour before the labeling incubation is complete, switch on the centrifugal evaporator and set the temperature to 75°C.

     
  11. 11.

    Standard ethanol precipitation: Spin down the labeled sample and reference tubes. Combine the labeled test and reference DNA samples by transferring the 25 μl of Cy5-labeled reference DNA to the 0.2 ml PCR tube containing the Cy3-labeled test sample. Add 25 μl COT Human DNA and 7.5 μl 3 M sodium acetate to each tube and vortex. Add 187.5 μl of absolute ethanol to each tube and invert two or three times to mix. Place tubes in a −80°C freezer and incubate for 10 min. Centrifuge the probes at 16,000  ×  g for 10 min and discard the supernatant. Add 500 μl of 70% ethanol, mix by inversion, and centrifuge at full speed for 5 min. Decant the supernatant. Keeping tube inverted, gently tap out any remaining droplets onto a folded tissue. Spin tubes in centrifuge briefly and remove the remaining ethanol with a micropipette. Allow the pellet to air-dry for 2 min at room temperature.

     
  12. 12.

    While the probes are being centrifuged to reduce the volume, preheat an aliquot of DS Hybridization Buffer at 75°C for 10 min. Make sure a sufficient volume of DS Hybridization Buffer is prepared, taking into account the viscosity of the liquid.

     
  13. 13.

    The microarrays are reasonably light resistant, but if problems with faint signals are encountered post-hybridization washes can be undertaken in the dark. Microarrays should be stored in a light-proof container. High levels of ozone can also lead to loss of fluorescence and can be a problem in regions with warm climates. Apparatus to remove ozone from the air are available.

     
  14. 14.

    It is important to include all the clones while minimizing the area around the microarray in order to reduce background pixels.

     
  15. 15.

    BlueFuse Multi provides fully automated analysis of all 24Sure experiments. This includes grid finding, signal estimation, normalization, exclusion of poor quality results, and combination of replicates, region detection, region classification, and region reporting. The microarray type is used to select algorithms optimized to the format of the microarray.

     
  16. 16.

    Gains of entire chromosomes should result in profile shifts (≥0.40–0.50) that are comparable to the profile shift observed for chromosome X. It is important to compare the shifts to chromosome X in order to determine whether it is a gain of a chromatid or an entire chromosome.

     
  17. 17.

    Losses of entire chromosomes should result in profile shifts to −0.90 and lower that are comparable to the profile shift observed for chromosome Y. Generally, loss of chromosomal material results in more dramatic shifts than gain. It is important to compare those shifts to chromosome Y in order to determine whether it is a loss of a chromatid or an entire chromosome.

     

Notes

Acknowledgement

D.W. is funded by the NIHR Biomedical Research Centre, Oxford.

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Reprogenetics UK, Institute of Reproductive SciencesOxfordUK
  2. 2.Nuffield Department of Obstetrics and Gynaecology, Women’s Centre John Radcliffe HospitalUniversity of OxfordOxfordUK

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