diagnosis: OPML patient id: Pt214B histology: Non dysplastic smoke_2_classes: S single_multiple_lesions: SG dna_index: 1 number_aneuploid_populations: 0 gender: M dapi_staining: Y sorting: N type_sample: CURETTE age_diagnosis: 66
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted using ArchivePure DNA kit (5-Prime Hamburg, Germany) with some modifications: after proteins precipitation the DNA was purified by phenol-chloroform extraction and collected by ethanol precipitation adding 20 μg of glycogen (20 mg/ml) as carrier. DNA quality and quantity were assessed by agarose gel and ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). A pool of normal male or female DNA (Promega, Madison, WI) was used as reference DNA. A quantity of 50 ng of genomic DNAs (test and reference) was amplified by GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO) that allows to generate a representative amplification of genomic DNA. The kit uses a linker mediated primer PCR amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR-amplifiable OmniPlex Library molecules flanked by universal priming sites. The OmniPlex library is then PCR amplified using universal oligonucleotide primers. After purification of PCR products by GenElute PCR Clean-Up Kit (Sigma-Aldrich), amplified DNA was quantified using the ND-1000 Spectrophotometer (Thermo Scientific).
Label
Cy5
Label protocol
For 2x105k arrays: For each array, 2 μg amplified test and reference DNAs were labeled by Bioprime Labeling Kit (Invitrogen, Paisley, UK) following the manufacture's protocol. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore, Billerica, MA) according to the manufacturer's protocol. Labeled DNA quality analysis and quantization were performed by NanoDrop ND-1000 Spectrophotometer and parameters that predicted successful hybridization were a specific activity of 25 to 40 and 20 to 35 for cyanine-3 and cyanine-5 labeled sample, respectively. Prior to the denaturation step at 95°C for 3 min, 50 μg Cot-1 DNA (Invitrogen) and control targets (Agilent Technologies) were added to cyanine 5- and cyanine 3-labeled DNA mixture. For 4x180k arrays: 0.8 µg of amplified test and reference (female or male normal genomic DNA: Promega, Madison,WI) were labeled by Sure Tag DNA labeling kit (Agilent Technologies) with Cy5-dUTP and Cy3-dUTP respectively according to CGH Enzymatic Labeling Kit Protocol v.7.2 (Agilent Technologies). Unicorporated nucleotides were then removed using Centrifugal filters (Amicon Ultra 0.5ml, Millipore) according to manifacturer’s protocol. Quality analysis and quantization of labeled DNA were performed by (Nanodrop ND-1000 Spectrophotometry) measuring A260 (for DNA), A550 (for Cy5) and A649 (for Cy3) to evaluated yield, degree of labeling or specific activity: parameters that predicted successful of hybridisation.
Channel 2
Source name
Commercially available normal genomic DNA, gender matched
DNA was extracted using ArchivePure DNA kit (5-Prime Hamburg, Germany) with some modifications: after proteins precipitation the DNA was purified by phenol-chloroform extraction and collected by ethanol precipitation adding 20 μg of glycogen (20 mg/ml) as carrier. DNA quality and quantity were assessed by agarose gel and ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). A pool of normal male or female DNA (Promega, Madison, WI) was used as reference DNA. A quantity of 50 ng of genomic DNAs (test and reference) was amplified by GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO) that allows to generate a representative amplification of genomic DNA. The kit uses a linker mediated primer PCR amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR-amplifiable OmniPlex Library molecules flanked by universal priming sites. The OmniPlex library is then PCR amplified using universal oligonucleotide primers. After purification of PCR products by GenElute PCR Clean-Up Kit (Sigma-Aldrich), amplified DNA was quantified using the ND-1000 Spectrophotometer (Thermo Scientific).
Label
Cy3
Label protocol
For 2x105k arrays: For each array, 2 μg amplified test and reference DNAs were labeled by Bioprime Labeling Kit (Invitrogen, Paisley, UK) following the manufacture's protocol. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore, Billerica, MA) according to the manufacturer's protocol. Labeled DNA quality analysis and quantization were performed by NanoDrop ND-1000 Spectrophotometer and parameters that predicted successful hybridization were a specific activity of 25 to 40 and 20 to 35 for cyanine-3 and cyanine-5 labeled sample, respectively. Prior to the denaturation step at 95°C for 3 min, 50 μg Cot-1 DNA (Invitrogen) and control targets (Agilent Technologies) were added to cyanine 5- and cyanine 3-labeled DNA mixture. For 4x180k arrays: 0.8 µg of amplified test and reference (female or male normal genomic DNA: Promega, Madison,WI) were labeled by Sure Tag DNA labeling kit (Agilent Technologies) with Cy5-dUTP and Cy3-dUTP respectively according to CGH Enzymatic Labeling Kit Protocol v.7.2 (Agilent Technologies). Unicorporated nucleotides were then removed using Centrifugal filters (Amicon Ultra 0.5ml, Millipore) according to manifacturer’s protocol. Quality analysis and quantization of labeled DNA were performed by (Nanodrop ND-1000 Spectrophotometry) measuring A260 (for DNA), A550 (for Cy5) and A649 (for Cy3) to evaluated yield, degree of labeling or specific activity: parameters that predicted successful of hybridisation.
Hybridization protocol
For 2x105k arrays: Hybridization mix was incubated at 37°C for 30 min, and applied to array. Hybridization was carried out for 40 hr at 65°C in a rotating hybridization oven (Agilent Technologies) at 20 rpm. For 4x180k arrays: Cy5-Labeled tumour DNA was combined with an equivalent amount of Cy3-labeled reference DNA. Repetitive sequences were blocked with human Cot-1 DNA (Invitrogen) and samples were hybridised with Oligo aCGH/ChIP-on-chip Hybridization Kit onto SurePrint G3 Human CGH Microarrays, 4x180K (Agilent) according to manufacturer’s instructions. Following hybridization at 65°C for 24 hours in a rotating hybridization oven (Agilent Technologies) at 20rpm. Microarray slides were washed according to manufacturer’s instructions
Scan protocol
For 2x105k arrays: spot fluorescence was measured by the Agilent G2565BA Scanner and images were processed by Feature Extraction software (version 9.5.3.1) (Agilent Technologies). For 4x105k arrays: slides were scanned immediately at 3 microns using the Agilent G2505C Scanner and images were processed by Feature Extraction software v11.01.1 (Agilent Technologies).
Data processing
Log10 ratios from Agilent feature extraction files were imported in R, averaged over replicates and log2 transformed with the limma package [Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W and Smyth GK (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research, 43.]. Common probes among the two microarray designs were retained for downstream analysis, and probes mapping on chromosome Y were discarded, resulting in the 87,891 probes reported in the matrix template.
Genomic DNA damage in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs). Early detection of DNA aneuploidy and chromosomal aberrations in non dysplastic OPMDs.
Data table header descriptions
ID_REF
VALUE
Log2 ratio; probe replicates are averaged in the data matrix
