tissue: spleen disease state: Splenic marginal zone lymphomas
Extracted molecule
genomic DNA
Extraction protocol
The DNA was extracted using the standard phenol-chloroform method. All DNA was quantified using the Nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE USA). DNA quality was assessed by the 260:280 ratio and its integrity by agarose gel ethidium bromide visualization.
Label
Cy5
Label protocol
2 μg of unamplified genomic DNA (tumour DNA) was digested with DpnII restriction enzyme (New England Biolabs, Beverly, MA) and labelled using random primers (BioPrime Labeling System, Invitrogen, Carlsbad, Calif) and Cy5-dCTP (CyDyeTM 5-dCTP, Amersham Biosciences, Piscataway, NJ) fluorescent dye for paired hybridization samples. The incorporation of labelled nucleotide was quantified using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).
disease state: healthy sample type: human placenta of healthy donors tissue: placenta
Extracted molecule
genomic DNA
Extraction protocol
The DNA was extracted using the standard phenol-chloroform method. All DNA was quantified using the Nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE USA). DNA quality was assessed by the 260:280 ratio and its integrity by agarose gel ethidium bromide visualization.
Label
Cy3
Label protocol
2 μg of unamplified genomic DNA reference material (placental DNA) was digested with DpnII restriction enzyme (New England Biolabs, Beverly, MA) and labelled using random primers (BioPrime Labeling System, Invitrogen, Carlsbad, Calif) and Cy3-dCTP (CyDyeTM 3-dCTP Amersham Biosciences, Piscataway, NJ) fluorescent dye for paired hybridization samples. The incorporation of labelled nucleotide was quantified using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).
Hybridization protocol
Labelled test and reference DNA samples were mixed equitably, co-precipitated in the presence of Cot-1 human DNA (Roche, Indianapolis, IN) with ethanol, washed, and resuspended in hybridization solution (50% formamide, 10% Dextran sulphate, 2X standard saline citrate, 10mM Tris pH 7.6, 2.7% sodium dodecyl sulphate and 10μg/μl of yeast tRNA). DNA mixtures were cohybridized to the arrays in a GENETAC (Genomic Solutions, Ann Arbor, Michigan, USA) for 48 hours at 42ºC in accordance with the manufacturer’s recommended protocol.
Scan protocol
Images and signal intensities were acquired using a GenePix 4000B (Axon Instruments, Burlingame, CA) dual laser scanner in combination with GenePix Pro4.0 (Axon Instruments, Burlingame, CA) imaging software.
Data processing
Fluorescence ratios were normalized using the median of the fluorescence ratios of every spot, computed as log2 values. The log2 ratio of each clone was normalized to the median log2 ratio of the 20 control hybridizations, after which the median of the three spots was calculated.
