Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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DNA and RNA analysis of blood and muscle from bodies with variable postmortem intervals.

Author(s): Hansen J, Lesnikova I, Funder AM, Banner J

Publication: Forensic Sci Med Pathol, 2014, Vol. 10, Page 322-8

PubMed ID: 24913557 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine whether postmortem interval (PMI) affects DNA and RNA yields or impedes PCR-based analysis of skeletal muscle and blood specimens. Muscle specimens were collected from the psoas major muscle of 45 individuals following postmortem delays that ranged between 1 and 14 d or longer then preserved by freezing at -80°C or by formalin-fixation, paraffin embedding.  Blood specimens, which could only be collected via the femoral vein from 28 individuals due to coagulation and hemolysis when PMI was > 14 d, was frozen at -80°C.  Preserved specimens were stored for 15-22 months prior to DNA and RNA extraction.  DNA was extracted from blood using the Qiagen Blood DNA isolation kit and from muscle specimens using the nucleospin tissue DNA kit alone for frozen specimens and following xylene deparaffinization and proteinase K digestion for FFPE specimens (four 20 µm-thick FFPE sections were used for extraction). RNA was extracted from frozen muscle specimens using Trizol, while FFPE specimens (two 20 µm-thick FFPE sections) underwent xylene deparaffinization, proteinase K digestion, and extraction using Roche's High PURE RNA paraffin kit. Statistical analysis was not performed.

   Summary of Findings:

   Although blood collection was not possible in the majority of cases when PMI was > 14 d (8/9), PCR amplification of a 200 bp DNA fragment was possible for all 28 blood specimens collected, regardless of PMI.  PCR amplification of DNA extracted from skeletal muscle specimens was also successful in the majority of cases for both frozen (89-100%) and FFPE (78-100%) specimens. However, the mean amplifiable fragment length was lower when PMI was ≥ 3 d compared to shorter PMI for both FFPE (median 400 vs. 1000 bp) and frozen specimens (median 600 vs 1000 bp). DNA yield also declined with advancing PMI for both frozen and FFPE muscle specimens, while yields were more variable among blood specimens.  Similar results were obtained for RNA, with yields declining as PMI increased for both FFPE and frozen specimens. However, the success rate of qRT-PCR amplification of a 86 bp fragment using RNA extracted from frozen and FFPE muscle specimens declined with advancing PMI from 100% after a PMI of 1-3 d to 78% (frozen) or 33% (FFPE) after a PMI of 3-7 d, and 33% (frozen) and 11% (FFPE) after a PMI > 14 d. The Cq range increased with advancing PMI from 25-35 after a PMI of 1-3 d to 33-39 after a PMI of 3-7 d in frozen specimens but not FFPE specimens.

   Biospecimens

   • Tissue - Muscle (Skeletal)
   • Bodily Fluid - Blood

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Not specified
   • Autopsy

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	1-3 d 3-7 d 7-14 d >14 d
   PCR Specific	Targeted nucleic acid	PYGM
   PCR Specific	Length of gene fragment	200 bp 400 bp 600 bp 800 bp 1000 bp
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACADM

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