Computational protocol: Deletions of multidrug resistance gene loci in breast cancer leads to the down-regulation of its expression and predict tumor response to neoadjuvant chemotherapy

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Protocol publication

[…] To study CNVs of MDR gene loci, microarray analysis was performed using high density microarray platform Affymetrix (USA) CytoScan™ D Array, (http://www.affymetrix.com/esearch/search.jsp?pd=prod520004&N=4294967292). The array contained 2.67 million markers, 1.9 million non-polymorphic markers and more than 750 000 single nucleotide polymorphism (SNP) markers that allowed structural variations of more than 36 000 genes to be determined. The presence of SNP markers on the microarray enabled copy number analysis to detect gains and losses in the DNA and loss of heterozygosity. Procedures of sample preparation, hybridization and scanning were performed in accordance with the manufacturer's protocol using the system Affymetrix GeneChip® Scanner 3000 7G (Affymetrix, USA). The Chromosome Analysis Suite 2.0 software (Affymetrix, USA), which is specifically devised for analyzing microarray results from the CytoScan™ HD Array, was used. Unbalanced chromosomal aberrations (deletions and amplifications, or Loss and Gain) were detected in chromosomal regions 3q27.1, 4q22.1, 6p21.3, 7q21.12, 10q24.2, 16p13.11, 16p11.2 and 21q22.3. Because stromal elements and other normal cells were presented in tumor tissue samples, the percentage of normal genomic DNA was high in the obtained DNA. The Microchip CytoScan™ HD Array can detect at least 5% of mutant DNA. In nearly all cases, CNV were mosaic, i.e., mutant tumor DNA was detected along with the normal DNA. Additionally, the percentage of mutant DNA copy number state (CN-state) ranged from 15 to 88%. The Chromosome Analysis Suite 2.0 software graphically presents mosaicism as allele peaks with 4 bands (AAA, AAB, ABB, BBB). The snpQC value ranged from 13 to 25 and negatively correlated with the CNV frequency. Even if the snpQC values were low, the width of the allele peaks was “good” (according to the progenetix resource []) for CNV identification. [...] The presence of structural CNV in MDR gene loci was assessed using the Chromosome Analysis Suite 2.0 software. Statistical analyses were performed using the STATISTICA 8.0 software (StatSoft, Tulsa, OK, USA). The arithmetic mean value and standard error were calculated for each sample group, and Mann-Whitney U-test was applied to identify the link between the expression levels of MDR genes in breast tumors and the presence/absence of deletion in the MDR loci. A two-sided p-value was calculated using Fisher's exact test http://vassarstats.net/odds2x2.html, and the Bonferroni correction was applied to address the problem of multiple comparisons and was calculated as the each p-value multiplied by the number of comparisons []. […]

Pipeline specifications

Software tools SNPQC, Statistica, VassarStats
Databases Progenetix
Applications Miscellaneous, SNP array data analysis
Organisms Homo sapiens, Martes pennanti
Diseases Breast Neoplasms, Huntington Disease, Neoplasms