DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: The effect of formalin fixation and paraffin embedding.
Author(s): Funabashi KS, Barcelos D, Visoná I, E Silva MS, E Sousa ML, de Franco MF, Iwamura ES
Publication: Pathol Res Pract, 2012, Vol. 208(10), Page 584-91
PubMed ID: 22920941 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of tissue type, specimen preservation, storage of formalin-fixed paraffin-embedded (FFPE) specimens and DNA extraction method on DNA yield and purity, PCR success rates and short tandem repeat (STR) analysis from specimens obtained at autopsy.
Conclusion of Paper
DNA yield was highest and the purity was most consistent when extraction was completed with phenol-chloroform. PCR was successful for all frozen specimens, regardless of DNA extraction method or targeted gene, but the rate of amplification success in FFPE specimens decreased with increasing storage duration and was dependent on targeted gene and extraction method. Using recent FFPE specimens, only DNA extracted with the QIAamp kit had 100% PCR success. STR analysis was not possible with most FFPE specimens, but when it was possible, results were most often obtained using DNA extracted with the QIAamp kit from spleen or liver tissues as opposed to brain tissue or DNA extracted with phenol-chloroform.
Studies
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Study Purpose
The purpose of this study was to determine the effects of tissue type, preservation method, storage of FFPE specimens and DNA extraction method on DNA yield and purity, PCR success rates and STR analysis. Liver, spleen, and brain specimens were obtained after a postmortem interval of 8-24 h. DNA was extracted from two 10 uM xylene-deparaffinized FFPE sections or 25 mg of frozen tissue. Prior to the phenol-chloroform and salting out extraction procedures, specimens were digested with proteinase K overnight.
Summary of Findings:
DNA yield was highest and the purity was most consistent when extraction was completed with phenol-chloroform. PCR was successful for all frozen specimens, regardless of DNA extraction method or gene target. The rate of amplification success in FFPE specimens decreased with increasing storage duration and was dependent on gene target and extraction method. Using FFPE specimens stored for less than 1 year, only DNA extracted with the QIAamp kit yielded a 100% PCR success rate. However, the highest amplification success rates from FFPE specimens stored for 1 or 5 years were obtained using DNA extracted by salting-out, but background amplification was also high. Amelogenin was not amplifiable from any stored FFPE block, regardless of DNA extraction method, or storage length. STR analysis was not possible with most FFPE specimens, but when it was possible, results were most often obtained using DNA extracted from spleen or liver tissues with the QIAamp kit as opposed to brain tissue or DNA extracted with phenol-chloroform. Amplification of amelogenin was more successful from brains fixed for 7 days in formalin and processed in ethanol washes and xylol than from brain specimens fixed for 24 h and not further processed.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Autopsy
- Not specified
Platform:
Analyte Technology Platform DNA PCR DNA Microsatellite analysis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Biospecimen Acquisition Biospecimen location Brain
Liver
Spleen
Analyte Extraction and Purification Analyte isolation method Phenol chloroform extraction
QIAamp mini kit
Salting-out
Storage Storage duration 0 years
1 year
5 years
Biospecimen Preservation Time in fixative 24 h
7 days
PCR Specific Targeted nucleic acid Beta-actin 136
Amelogenin (212-218 bp/106-112 bp)