Biomedical Engineering Reference
In-Depth Information
Commercial oligonucleotide aCGH platforms include Illumina (60 mer), Operon
(70 mer), Affymetrix (25 mer), Agilent (60 mer) and NimbleGen (45-85 bp), the latter with
now up to 2.1 million oligonucleotides on the array [11]. The quality of the oligonucleotide
aCGH platforms is rapidly improving, with single oligonucleotides rapidly reaching the
sensitivity of single BAC clones. Not all of the current oligonucleotide aCGH platforms
can make a definite call for loss or gain using a single oligonucleotide, but in some
cases three to five adjacent oligonucleotides are necessary for a reliable call [6, 11].
Moreover, owing to improvements in protocols, DNA isolated from FFPE tumor samples
now works comparable to DNA from fresh material (Protocols 1.1-1.4) on long (
>
50 bp)
oligonucleotide arrays (see Figure 1.1).
The principle of oligonucleotide aCGH is the same as all aCGH variants: Labeled tumor
DNA (Protocol 1.5) competes with differentially labeled normal DNA for hybridizing to
an array of oligonucleotides (Protocol 1.6). Using a specialized scanner and digital image
processing software, the ratio of the two is measured per spot on the array. Deviations
from the normal ratio of 1.0, or the log
2
ratio of 0.0, represent a copy number aberration
of genetic material in the tumor. The final result is DNA copy number information for
all the oligonucleotides on the array, which can be ordered according to the chromosomal
localization (Protocol 1.7). Graphics may represent all spots at once or only those belonging
to one chromosome (see Figure 1.1). The sensitivity of CGH depends on the purity of the
tumor sample and of the quality of the DNA obtained.
The oligonucleotide aCGH protocol presented provides a highly sensitive and repro-
ducible platform applicable to DNA isolated from both fresh and FFPE tissue. We do
not present protocols on the preparation of the array slides, since these can be purchased
commercially.
PROTOCOL 1.1 DNA Extraction from Fresh or Frozen Tissue
Equipment and reagents
•
Wizard Genomic DNA Purification Kit (Promega, A1120), containing:
—EDTA/nuclei lysis solution
—proteinaseK(20mg/ml)
—RNase solution (100mg/ml)
—protein precipitation solution
Phase lock gel (PLG, Eppendorf)
•
Phenol solution (e.g. Sigma-Aldrich, P-4557)
•
Chloroform
•
•
Isopropanol
•
Phenol/chloroform: 50% (v/v) phenol, 50% (v) chloroform
•
TE: 10 m
M
Tris-HCl, pH 8.0, 1m
M
EDTA
•
Ethanol (70% (v/v) and 100% (v/v), both ice-cold)
•
Sodium acetate (3
M
,pH5.2).
