Project description:Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of the present study is to compare aCGH platforms suitable for copy number analysis using FFPE derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP based platform (Affymetrix), were evaluated using seven FFPE colon cancer samples, median absolute deviation (MAD), deflection, signal-to-noise ratio (SNR) and DNA input requirements were used as quality criteria. Large differences were observed in MAD values and deflection between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared to Affymetrix (0.22). Contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. Since the deflection compensates for the MAD the Signal to Noise Ratios (SNR) were comparable; Agilent ranks first, Affymetrix second and NimbleGen third with SNRs of 3.9, 3.6 and 3.3 respectively. DNA input amounts of 40ng are sufficient for high quality profiles with Affymetrix. For Agilent DNA input amounts of 50ng are sufficient for high quality profiles. For results of similar quality NimbleGen requires at least 100ng. Copy number analysis using DNA derived from FFPE archival material is feasible and shows reproducible results on high-resolution copy number platforms. Input amounts of DNA from FFPE material lower than recommended still yield high quality profiles without additional amplification steps. Overall study comprises 6 samples analyzed across three platforms with an additional dilution range (5 concentrations) of a seventh sample on two platforms. This Series represents NimbleGen platform-derived data.

Project description:Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of the present study is to compare aCGH platforms suitable for copy number analysis using FFPE derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP based platform (Affymetrix), were evaluated using seven FFPE colon cancer samples, median absolute deviation (MAD), deflection, signal-to-noise ratio (SNR) and DNA input requirements were used as quality criteria. Large differences were observed in MAD values and deflection between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared to Affymetrix (0.22). Contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. Since the deflection compensates for the MAD the Signal to Noise Ratios (SNR) were comparable; Agilent ranks first, Affymetrix second and NimbleGen third with SNRs of 3.9, 3.6 and 3.3 respectively. DNA input amounts of 40ng are sufficient for high quality profiles with Affymetrix. For Agilent DNA input amounts of 50ng are sufficient for high quality profiles. For results of similar quality NimbleGen requires at least 100ng. Copy number analysis using DNA derived from FFPE archival material is feasible and shows reproducible results on high-resolution copy number platforms. Input amounts of DNA from FFPE material lower than recommended still yield high quality profiles without additional amplification steps. Overall study comprises 6 samples analyzed across three platforms with an additional dilution range (5 concentrations) of a seventh sample on two platforms. This Series represents Agilent platform-derived data.

Project description:Soft tissue sarcomas (STS) often present a significant diagnostic challenge as many STS bear histologic resemblance, but are known to have very different clinical and biologic characteristics. Some STS subtypes are characterized by specific genetic abnormalities and this has helped in their classification, diagnosis and even treatment. However, a large majority of STS have no known specific genetic aberrations even though they almost always have highly aberrant karyotypes. We therefore hypothesize that the latter subgroup of STS bear genetic abnormalities that are sub-type specific, but as yet unidentified. High-resolution mapping of copy number aberrations in cancer genomes is a valuable way of identifying recurrent genomic changes that could be of pathogenetic significance. Traditionally, this has been done using high quality DNA obtained from fresh frozen tissue or cells and archived tissue is generally regarded as unsuitable because of the degradative effects of formalin fixation on DNA. Utility of archival tumour material for such molecular genetic analysis is vital, especially for rare cancers like STS but recent efforts to accomplish this have produced variable results. We therefore set out, in addition to optimize a protocol for obtaining genomic copy number data from formalin-fixed, paraffin-embedded (FFPE) STS material that is comparable to that from fresh frozen (FF) material. Microarray-based Comparative Genomic Hybridization (aCGH), a high- resolution, genome-wide method was used to identify somatic copy number aberrations (SCNAs) in primary STS samples (fresh frozen and archival FFPE), using an optimized protocol for labeling DNA. Findings were confirmed using Conventional Cytogenetics and Fluorescence in-situ Hybridization (FISH). Data obtained from paired samples (FF and FFPE) of the same tumours showed similar results and array results were consistently of good quality. On-going analysis of the recurrent SCNAs in combination with expression data and clinical correlates may serve to identify specific patterns that can serve as diagnostic markers, characterize subgroups with prognostic implication or identify potential therapeutic targets. To identify common CNAs among LMS fresh and FFPE. 25 samples in total: 22 individual FFPE cases; 3 cases also obtained fresh. Reference DNA was obtained from same patient when possible, otherwise commercial genomic DNA was used [Promega® UK with Cat Nos. G1471 (male) and G1521 (female)].

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Three commercial array platforms compared for DNA copy number detection with DNA isolated from FFPE tissue [NimbleGen]

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