Transcriptional profiling and genotyping of degraded nucleic acids from autopsy tissue samples after prolonged formalin fixation times - PubMed (original) (raw)
. 2011 Jan 6;4(2):156-61.
Affiliations
- PMID: 21326810
- PMCID: PMC3037201
Transcriptional profiling and genotyping of degraded nucleic acids from autopsy tissue samples after prolonged formalin fixation times
Antonio Ferruelo et al. Int J Clin Exp Pathol. 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-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-α 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.
Keywords: Autopsy; critical illness; formalin; gene expression; polymorphism; pre-amplification.
Figures
Figure 1
Recovery and integrity of isolated nucleic acid from FFPE tissues. A. Extraction yield and quality of nucleic acids from FFPE liver tissue samples. B. RIN and size distribution of RNA, according to formalin fixation time. Group 1, less than 15 days; group 2, 60-90 days; group 3, 150-180 days; group 4, 240-270 days. Lane L contains molecular weight marker RNA
Figure 2
A. Effect of pre-amplification on TaqMan gene expression pattern using qRTPCR in FFPE tissues. The pre-amplification (open bars) generated lower Ct levels in comparison with no pre-amplification (solid bars) in all groups. B. Electrophoretic pattern of DNA on 1% agarose ethydium bromide-stained gels photographed under ultraviolet light. C. Ethydium-bromide-stained gel showing samples of the ACE genotypes on 2% agarose gels. The longer fragment (84 bp) corresponds to the deletion (D) allele; the shorter fragment (65 bp), to the insertion (I) allele. Groups according to fixation times (as in Figure 1). M: marker (1kb DNA ladder), M1: marker (1000 pb DNA ladder) and C+ (positive control).
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