59º Congresso Brasileiro de Genética Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013 Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil www.sbg.org.br - ISBN 978-85-89109-06-2 140 CMA TROUBLESHOOTING: SAMPLE REFERENCES FOR DNA FIXED IN CARNOY SOLUTION da Cruz AS1,2,, Pinto IP1, Cunha DMC1, Franco FC1, Silva DM1,3, da Silva CC1,4, da Cruz AD1,4 Núcleo de Pesquisas Replicon, Pontifícia Universidade Católica de Goiás; 2Programa de Pós Graduação em Biologia, Universidade Federal de Goiás; 3Instituto de Ciências Biológicas, Universidade Federal de Goiás; 4Laboratório de Citogenética e Genética Molecular Humana (LaGene), Secretaria da Saúde do Estado de Goiás. 1 [email protected] Keywords: Cytogenetics, lymphocyte culture, Carnoy solution, Microarray, CMA. Rapid developments in molecular cytogenetics was provided by identifying control references to be able predictive for utilization of the Chromosomal Microarray Analysis (CMA). Nowadays, the CMA has been conducted mainly in fresh, frozen and paraffin tissue. The Affimetrix CMA provides optimum coverage and great perspectives for use in research laboratories, in an attempt to resolve cases of unexplained syndromes with the use of classical cytogenetics. However, there are difficulties in the use of the CMA in samples preserved in Carnoy’s solution. The disadvantage of this process is that the samples preserved in Carnoy’s solution may not be range values of reference for samples already existing. The aim of this study was to evaluate the use of the CMA in samples fixed in Carnoy’s solution. We evaluated two samples of individuals preserved by ≥ 5 years. Samples of peripheral blood were obtained by venipuncture and the realization of cell culture was performed according to the protocol of T lymphocyte established by the LaGene (SES-GO) laboratory. After the procedure of the T lymphocyte culture, samples were fixed in Carnoy’s solution 3:1 (methanol/ acetic acid) and stored in refrigerator. These samples were sent for DNA extraction by using QIAamp DNA Mini Kit (Qiagen, USA) and quantitated by NanoVue TM Plus (GE Healthcare, USA) according to the manufacturer’s suggested protocol. After extraction and quantification, samples were genotyped in a CytoScan® HD (Affymetrix®). The microarray used in this study was the GeneChip® HD (750,000 SNPs). The CMA was performed according to the manufacturer’s suggested protocol. The results of CMA are referenced by the quality controls suggested by Affymetrix ®: snpQC >15.000, mapd <0.25 and waviness <0.12, to ensure a read reliability of GeneChip® HD. The reference values are generated by the use of software Chromosome Analysis Suite (Chas) 2.0 (provided by Affymetrix®, USA). The reference values presented in this study were snpQC 10.218, mapd 0.283 and waviness 0.177 to sample 1, and snpQC 6.264, mapd 0.282 and waviness 0.176 to sample 2. The CMA performed with fixed samples in Carnoy’s solution did not presented necessary quality to be validated by Chas and this prevent its examination by the software. Thus, in order to enable these samples were validated by Chas program, they were referenced with controls of samples that had DNA extracted from paraffin material (provided by Affymetrix®). Thus, it was possible to perform the reading and interpretation of the data generated by the GeneChip® HD. With these results it was possible to conclude that the application of CMA in samples fixed in Carnoy’s solution was possible by the use of reference values for paraffin samples. This step was essential to the validation and reading of GeneChip® HD by the software of analysis Chas. Financial Support: CNPq and Fapeg