Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Effect of Postmortem Interval and Years in Storage on RNA Quality of Tissue at a Repository of the NIH NeuroBioBank.

Author(s): White K, Yang P, Li L, Farshori A, Medina AE, Zielke HR

Publication: Biopreserv Biobank, 2018, Vol. , Page

PubMed ID: 29498539 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RIN values in 10 different tissue types and investigated the effects of PMI, storage duration, patient age, pH, and diagnosis on the RIN of RNA from cortical and cerebellum specimens. The effects of PMI and storage duration on real-time PCR amplification of 10 genes in RNA with a RIN or 7.5 and 8.5, respectively, were also examined. At autopsy (PMI), half of the brain from 1068 patients was frozen in isopentane-dry ice and stored at -80˚C in heat-sealed bags. Total RNA was extracted from 60-80 mg of homogenized prefrontal cortex and cerebellum using the RNeasy Lipid Tissue kit. RNA was quantified by NanoDrop spectrometry and integrity was investigated using a bioanalyzer. The effect of RNA integrity and storage duration on gene expression as determined by real-time PCR were each investigated using 12 specimens and the effects of PMI on gene expression measured by real-time PCR was investigated using 15 specimens. The targets of real-time PCR were chosen based on their turnover and included three genes with a half-life of 4 h (PAK2, SERBP1, and alpha tubulin), three genes with a half-life of 8 h (aconitase 1, NAPA, PRDX5), and four that are considered stable (ETFB, GSTM5, MCTS1, ACTB).  

   Summary of Findings:

   Compared to cerebellum, lower RIN values were found in psoas muscle (P=0.009), heart (P=0.003), testis (P=0.007), liver (P<0.001), lung (P<0.001), kidney (P<0.001), ileum (P<0.001), and spleen (P<0.001). Using specimens from all 1068 donors, RIN values of cortex and cerebellum specimens were modestly correlated (R²=0.46, P<0.001) and were greater than 6 for more than 80% of specimens. RIN was significantly, very weakly inversely correlated with PMI in cortical (R²=0.013, P<0.001) specimens and trended a very weak inverse correlation with PMI in cerebellum (R²=0.003, P=0.06). Further inspection showed RIN to be comparable in specimens with a PMI of 0-6 h, 7-12 h, 13-24 h, and 25-36 h; but lower in specimens with a PMI of 37-48 h or more than 49 h (P>0.05, both). RIN was only very weakly correlated with frozen storage duration (R²=0.02 and P<0.001 in both cortical and cerebellum specimens), patient age (R²=0.03 and P<0.001 in both cortical and cerebellum specimens), and pH (R²=0.006 and P=0.03 in cortical specimens, and R²=0.007 and P=0.024 in cerebellum specimens). RIN values were lower for specimens from patients with Alzheimer’s than all others and pH was higher in patients with autism than in other patients.

   As expected, specimens with a low RIN had higher CT values than those with higher RIN, and this was found regardless of the target. In specimens with a RIN of 7.5, PMI had no effect on the CT values of the 10 genes. In specimens with a RIN of 8.5, years in storage (1-23 years) did not affect CT values for 9 of 10 genes but NAPA expression increased with increasing storage duration (correlation not determined).

   Biospecimens

   • Tissue - Brain
   • Tissue - Muscle (Skeletal)
   • Tissue - Heart
   • Tissue - Liver
   • Tissue - Small Bowel
   • Tissue - Testis
   • Tissue - Spleen
   • Tissue - Kidney
   • Tissue - Lung

   Preservative Types

   • Frozen

   Diagnoses:

   • Alzheimer's Disease
   • Amyotrophic Lateral Sclerosis
   • Other diagnoses
   • Epilepsy
   • Parkinson's Disease
   • Depression

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	0-100 years
   Preaquisition	Diagnosis/ patient condition	Alzheimer’s disease Adrenoleukodystrophy Amytrophic lateral sclerosis Autism Depression Dystonia Epilepsy Parkinson’s disease Prader-Willi syndrome Other
   Preaquisition	Postmortem interval	0-6 h 7-12 h 13-24 h 25-36 h 37-48 h >49 h
   Biospecimen Aliquots and Components	pH	A range of values investigated
   Biospecimen Acquisition	Biospecimen location	Cerebellum Prefrontal cortex Psoas muscle Heart Testis Liver Lung Kidney Ileum Spleen
   Storage	Storage duration	0-23 years

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2. Study Purpose

   This study investigated the effects of storing cerebellum specimens at -80˚C for 0.5-1 year or 13-14 years on DNA integrity. Cerebellums from 16 patients were frozen in isopentane-dry ice and stored at -80˚C in heat-sealed bags for 0.5-1 year (5 specimens) and 13-14 years (11 specimens). DNA was extracted using the DNeasy Blood and Tissue Kit and quantified by NanoDrop spectrophotometer. DNA integrity was determined by electrophoresis.

   Summary of Findings:

   There was no effect of frozen storage duration on the integrity of DNA

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified
   • Autopsy

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0.5-1 year 13-14 years

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