Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program.
Author(s): Fan J, Khanin R, Sakamoto H, Zhong Y, Michael C, Pena D, Javier B, Wood LD, Iacobuzio-Donahue CA
Publication: Oncotarget, 2016, Vol. 7, Page 66906-66921
PubMed ID: 27602498 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of postmortem interval (PMI) on RNA recovery and integrity and DNA quality from normal, primary tumor, and metastatic tissues snap-frozen in liquid nitrogen. The effects of storage duration on RNA yield and integrity were also examined.
Conclusion of Paper
For normal tissue specimens, variability in RIN was tissue-type specific with the lowest RINs observed in kidney specimens and the highest in skeletal muscle specimens. Mean RINs did not differ significantly from normal and tumor tissues and there was no statistically significant correlation among RINs of matched liver and lung metastases. Variability in RINs was less in normal specimens with a PMI ≤ 5 h than for those with a PMI ≥ 21 h with strong negative correlations between RIN and PMI for liver and skin specimens but no correlations for heart or skeletal muscle. In contrast, there was no correlation between RIN and PMI in primary tumor tissues. The authors state that while higher overall RNA yields were found from tissue specimens stored ≤ 1 y compared to those stored long-term (>5 y) there was no correlation between RIN and storage duration. RNA sequencing was successful in all five pairs of matched normal and tumor pancreas specimens, regardless of RIN, and tissue type was easily determined between the two groups by the pattern of the top 250 genes differentially expressed. DNA damage was detected in four of the 36 specimens analyzed, all of which were from the liver and had concurrent RNA degradation and three of the four were from patients with a PMI ≥ 21 h.
Studies
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Study Purpose
This study investigated the effects of PMI on RNA quantity and integrity and DNA quality from normal, primary tumor, and metastatic tissues snap-frozen in liquid nitrogen. A total of 371 specimens from 80 patients were collected during autopsy. Normal (n=287) and primary tumor (n=52) tissue specimens from liver, lung, kidney, pancreas, spleen, heart, skeletal muscle, and skin and metastatic tissue specimens (16 liver and 16 lung) including 10 patient-matched primary-metastatic pairs were collected in cryovials, snap-frozen in liquid nitrogen, and stored at −80°C for 1 to <15 years. Specimens were categorized into one of four groups based on a PMI of ≤ 5 h, 6 – 10 h, 11 – 20 h, or ≥ 21 h. RNA was extracted from ~ 30 mg of tissue using RNeasy Mini Kits or Fibrous Tissue Mini Kits. RNA quantity was measured with a spectrophotometer and RNA integrity as indicated by RNA Integrity Number (RIN) was determined using a bioanalyzer. RNA from five matched pairs of normal and tumor pancreas tissues with RIN > 5 were further analyzed by next-generation sequencing (NGS). Genomic DNA was extracted using DNeasy Mini Kits from 36 specimens (7 normal liver, 9 normal kidney, 10 primary tumors, 5 liver metastases, and 5 lung metastases) collected from five patients with a PMI ≤ 5 h and from five patients with a PMI ≥ 21 h with a wide range of RIN values for each PMI group (2.3 - 9.4). DNA quantity was determined by fluorometry and quality was assessed by multiplex PCR after applying a two-step data normalization method and four amplicons that ranged between 132 and 295 bp in length.
Summary of Findings:
The overall mean RIN for all normal tissue specimens was 5.94 ± 2.5, although tissue-specific differences in RIN were observed, with the lowest RINs observed in kidney specimens (4.63 ± 1.95) and the highest in skeletal muscle specimens (9.01 ± 1.36). The overall mean RIN for all primary tumor specimens was 5.16 ± 2.4 and, of these, 83% (43/52) were pancreatic ductal adenocarcinomas (PDAs). Mean RINs did not differ significantly between PDA and normal pancreas tissue specimens (5.4 ± 2.4 versus 5.26 ± 2.56, respectively) or among the subset of 17 matched pairs of primary PDA and normal pancreas specimens (5.17 ± 2.35 versus 6.16 ± 1.96, respectively. Similarly, RINs of case-matched liver and lung metastases were not correlated with one another (r=0.3489, P=0.2025). Overall, lower RINs were observed among specimens with longer PMIs, although the magnitude of effect was tissue-dependent. Significant and strong negative correlations between RIN and PMI were observed for liver and skin specimens (r=-0.49, P=0.003 and r=-0.52, P=0.007, respectively), but not among heart or skeletal muscle (r=-0.14, P=0.451 and r=-0.03, P=0.866; respectively) or primary tumor tissues (r=-0.13, P=0.381). While specimens stored ≤ 1 y had higher overall RNA yields than those stored long-term (>5 y), storage duration was not correlated to RIN. Low RNA yields (defined as 20 µg/ml total RNA) were unrelated to PMI, histology, or the number of freeze/thaw cycles (data not provided). RNA sequencing was successful in all five pairs of case-matched normal and tumor pancreas specimens, regardless of RIN, and the two specimen types were easily differentiated from one another by differential expression patterns of 250 genes. No DNA damage was detected in 89% (32/36) of the specimens even though 17 of these 32 (53%) had RIN values less than 5. All four of the specimens with DNA damage were from the liver and had concurrent RNA degradation, three of which were from patients with a PMI ≥ 21 h. Two of these four specimens were degraded at all four of the loci analyzed and the remaining two specimens demonstrated only moderate damage of two of the four loci.
Biospecimens
- Tissue - Liver
- Tissue - Lung
- Tissue - Muscle (Skeletal)
- Tissue - Skin
- Tissue - Pancreas
- Tissue - Heart
- Tissue - Spleen
- Tissue - Kidney
Preservative Types
- Frozen
Diagnoses:
- Autopsy
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA PCR RNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Postmortem interval ≤ 5 h
6 – 10 h
11 – 20 h
≥ 21 h
Storage Storage duration < 1yr to >5 y
Biospecimen Acquisition Biospecimen location liver
lung
kidney
pancreas
spleen
heart
skeletal muscle
skin
Preaquisition Diagnosis/ patient condition Normal tissue
Primary tumor tissue
Metastatic tissue