Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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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

Conclusion of Paper

Studies

1. 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

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