Analysis of mRNA from human heart tissue and putative applications in forensic molecular pathology.
Author(s): Partemi S, Berne PM, Batlle M, Berruezo A, Mont L, Riuró H, Ortiz JT, Roig E, Pascali VL, Brugada R, Brugada J, Oliva A
Publication: Forensic Sci Int, 2010, Vol. 203, Page 99-105
PubMed ID: 20705404 PubMed Review Paper? No
Purpose of Paper
This paper investigated effects introduced by postmortem interval (PMI) on RNA yield and integrity and gene expression in heart specimens preserved by snap-freezing in liquid nitrogen (longer PMIs of 36-120 h) or immersion in RNAlater (shorter PMIs of 0.66-2.7 h). The potential effect of storage temperature during PMI (room temperature versus 1°C) was also examined among specimens procured after longer PMIs.
Conclusion of Paper
RNA integrity as determined by examination of 18S and 28S ribosomal RNA by bioanalyzer, gel electrophoresis, and microfluidic electrophoresis was considered acceptable for all seven organ donor specimens (PMI 0.66-2.7 h) but only for four of the nine autopsy specimens (PMI 36-120 h). RNA integrity numbers (RINs) were higher among specimens with intact RNA than specimens with degraded RNA as determined by gel and microfluidic electrophoresis. There were no differences in transcript levels of the reference gene GUSB or in COL1A1 and COL3A1 between autopsy and organ donor specimens but NOS3 levels differed significantly between the two groups of specimens. The authors state there were no statistically significant differences in RNA integrity as determined by gel and microfluidic electrophoresis in autopsy specimens stored at RT <12 h or >12 h after procurement or in specimens maintained at 1°C for ≤24 h or >24 h after procurement and no differences were observed in morphology between autopsy specimens regardless of PMI or cold ischemia time and temperature.
Studies
-
Study Purpose
This study investigated effects introduced by postmortem interval (PMI) on RNA yield and integrity and gene expression in heart specimens preserved by snap-freezing in liquid nitrogen (longer PMIs of 36-120 h) or immersion in RNAlater (shorter PMIs of 0.66-2.7 h). The potential effect of storage temperature during PMI (room temperature versus 1°C) was also examined among specimens procured after longer PMIs. Right ventricle specimens (~100 mg) from 9 individuals with normal cardiac function (PMIs ranging from 36-120 h) were obtained at autopsy, stored at room temperature (<12 h, 24 h, or 72 h) or 1°C (24 h, 48 h, 36 h, or 72 h), snap-frozen in liquid nitrogen, and then stored at -80°C. A portion of the specimens were formalin-fixed paraffin-embedded and 4 µm sections were stained with hematoxylin and eosin (H&E). Tissue specimens (5-10 mg) from seven organ donors (PMIs from 0.66-2.7 h) were obtained from hearts kept in a cardioplegic solution at 4°C (40-163 min), immersed in RNAlater, stored at 4°C for 24-72 h, and then RNAlater was removed before storage in liquid nitrogen until analysis. RNA was extracted with Trizol. RNA yields were determined spectrophotometrically, RNA integrity was assessed by bioanalyzer and agarose and microfluidic gel electrophoresis analysis of 18S and 28S ribosomal RNA (rRNA), and RINs assigned using a bioanalyzer. Gene expression of beta-glucuronidase (GUSB), Nitric Oxide Synthase-3 (NOS3), Collagen Type I Alpha 1 (COL1A1), and Collagen Type 3 Alpha 1 (COL3A1) was quantified by real-time qRT-PCR in autopsy specimens (n=4) and organ donor specimens (n=7).
Summary of Findings:
Integrity of total RNA as determined by bioanalyzer and gel electrophoresis of 18S and 28S ribosomal RNA bands differed significantly between specimens from organ donors snap-frozen in liquid nitrogen (PMI 0.66-2.7 h) and specimens obtained at autopsy and preserved in RNAlater (PMI 36-120 h) (P<0.05). RNA yield was comparable between autopsy specimens and specimens from organ donors (0.35 ± 0.02 µg vs. 0.26 ± 0.05 µg RNA/mg heart) and among autopsy specimens with either degraded RNA or intact RNA (0.37 ± 0.05 vs. 0.33 ± 0.05 µg RNA/mg heart). As expected, RINs were higher in autopsy specimens with intact RNA than specimens with degraded RNA (7.6 ± 0.21 vs. 4 ± 0.67, P<0.05). There were no differences in the expression levels of the reference gene GUSB or in COL1A1 and COL3A1 between autopsy and organ donor specimens but there were significant differences in NOS3 levels between the two groups of specimens (6.53 ± 0.86 ng-equiv. vs. 15.5 ± 3.33 ng-equiv., P<0.05). The authors state that autopsy specimens stored at RT <12 h after procurement tended to be less degraded than specimens maintained >12 h at RT but differences were not statistically significant and there were no differences in the number of autopsy specimens with degraded RNA when tissues were maintained at 1°C for ≤24 h or >24 h (data not provided). No differences were observed in morphology between autopsy specimens regardless of PMI or cold ischemia time and temperature.
Biospecimens
Preservative Types
- RNAlater
- Frozen
Diagnoses:
- Normal
- Autopsy
Platform:
Analyte Technology Platform Morphology H-and-E microscopy RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Spectrophotometry RNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Postmortem interval 0.66-2.7 h
36-120 h
Preaquisition Diagnosis/ patient condition autopsy
organ donor
Biospecimen Preservation Type of fixation/preservation Snap frozen
RNAlater
Storage Storage temperature Room Temperature
1°C
Storage Storage duration <12 h
> 12 h