Original Article Transcriptional profiling and genotyping of degraded nucleic acids from autopsy tissue samples after prolonged formalin fixation times
Antonio Ferruelo, Mariam El-Assar, José A Lorente, Nicolás Nin, Oscar Peñuelas, Pilar Fernández-Segoviano 2,3, Constancio Gonzalez, Andrés Esteban
Critical Care Department, Hospital Universitario de Getafe, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Spain; Pathology Department, Hospital Universitario de Getafe, Madrid, Spain; Department of Biochemistry, Molecular Biology and Physiology, and IBGM, Universidad de Valladolid and CSIC, Valladolid, Spain.
Received December 7, 2010; accepted January 5, 2011; Epub January 6, 2010; published February 1, 2011
Abstract: Background. Samples used for genotyping and transcription studies are obtained and conserved in very specific conditions. The possibility to use autopsy tissue samples, which contain nucleic acids of very poor quality, would open new possibilities for genetic studies. Methods. We have used liver tissue samples from autopsy cases to (i) determine its quality ; (ii) study gene expression of 13 genes involved in different cell processes, before and after cDNA pre-amplification (quantitative reverse transcriptase polymerase chain reaction); and (iii) analyze the presence of 2 common polymorphisms of relevance for illness (ACE I/D genotype by PCR amplification, and TNF-ALPHA-α promoter gene polymorphism, by DNA sequencing). Results. Samples were grouped according to different buffered formalin fixation times (group 1, <15 days; group 2, 60-90 days; group 3, 150-180 days; group 4, 240-270 days). Nucleic acids showed a time-dependent degradation. The expression of 13 genes could be studied in all cases from groups 1 and 2, only 7 from group 3 and none from group 4. cDNA preamplification allowed the study of all genes in all samples. DNA genotyping for ACE and TNF-ALPHAa promoter region was possible in all cases. Conclusions. We conclude that nucleic acids extracted from autopsy specimens after prolonged periods of time in formalin were of sufficient quality to study gene expression and genotyping using currently available methodology and cDNA pre-amplification. (IJCEP1012002).