Biomedical Engineering Reference
In-Depth Information
3 Examine the electropherogram patterns visually.
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4 Separate probe signals from background signals by automatic binning and data
filtering.
5 Quantify and normalize the probe signals (peaks).
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6 Chart DNA copy number alterations according to genomic locations.
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Notes
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MLPA product fragment lists should have the following plot settings - Genescan (ABI): dye,
time, peak length, peak height, peak area; Genemapper (ABI): peak dye, sample file name, peak
height, peak area.
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The protocol describes the creation of copy number ratio from MLPA product fragment lists
using MLPA-DAT.
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Reference runs are usually MLPA runs performed on normal human DNA samples within the
same experiment as the samples. Multiple reference runs are recommended for probability
calculation of the sample ratios found. Mean or median normalization are only recommended
with larger sample numbers investigating syndromes with a low prevalence.
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The DNA concentration, DNA ligation check and signal count are performed automatically.
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Quantification is fully automated in MLPA-DAT and includes removal of individual run variance.
The principle of normalization is twofold. First, all peak areas are converted into relative peak
areas by dividing each by the sum of all peak areas. Second, a normalization factor is calculated
from the ratio between the relative peak areas of all control probes of a sample and a reference
DNA. Normalization settings may depend on the MLPA kit and type of sample analysed; for
samples showing few aberrations, normalization can be done using all probes, but for samples
with multiple chromosomal aberrations, such as tumor samples, it is recommended to use the
control probes only. Control probe normalization sets the internal run normalization factor on
a number of MLPA-probes known to remain constant in the expected sample types, while the
amount of run variance is either determined on these probes only, or on all probes.
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This is automatic in MLPA-DAT.
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A deletion of one copy of a probe target sequence will usually be apparent by a reduction in
relative peak area for that probe amplification product of 35-55%. A gain in copy number from
two to three copies/diploid genome will usually be apparent by an increase in relative peak
area between 30 and 55%.
1.3 Troubleshooting
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Low concentration of DNA isolated from FFPE tissue
: When a low yield of DNA is
expected from FFPE tissue, glycogen can be added as a co-precipitant prior to ethanol
precipitation. This has no noticeable effect on aCGH results.
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DNA contaminants in MLPA
: MLPA reactions are more sensitive to contaminants than
ordinary PCR reactions. DNA samples extracted from FFPE tissues often contain contam-
inants. Small remnants of phenol may act as PCR inhibitors, giving amplification products
with lower average peak areas. Reducing the amount of DNA used will therefore some-
times have a beneficial effect. Normalization of these DNA samples should be performed
against DNA extracted from reference DNA of the same source of tissue.
