Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Effects of antemortem and postmortem variables on human brain mRNA quality: a BrainNet Europe study.

Author(s): Durrenberger PF, Fernando S, Kashefi SN, Ferrer I, Hauw JJ, Seilhean D, Smith C, Walker R, Al-Sarraj S, Troakes C, Palkovits M, Kasztner M, Huitinga I, Arzberger T, Dexter DT, Kretzschmar H, Reynolds R

Publication: J Neuropathol Exp Neurol, 2010, Vol. 69, Page 70-81

PubMed ID: 20010301 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of preacquisition (patient condition, age, and gender), acquisition (month of death, postmortem interval, and delay to preservation), preservation (freezing method) and storage variables (storage duration and transport temperature) on RNA integrity and expression in human brain specimens. This study included a total of 193 snap-frozen postmortem brains from the BrainNet Europe Biobank including specimens from patients with Alzheimer’s disease (12 patients), amyotrophic lateral sclerosis (11 patients), multiple sclerosis (66 patients), Parkinson’s disease (24 patients), Guillain-Barre syndrome (1 patient), multiple systems atrophy (3 patients), stroke (2 patients), progressive supranuclear palsy (2 patients), or no neurological conditions (72 patients). RNA quality of different anatomical brain regions and specimens from patients who experienced different antemortem events were also compared in 10 and 64 specimens, respectively. RNA was extracted by RNeasy tissue lipid mini kit.

   Summary of Findings:

   While RINs of the 193 extracted specimens ranged between 2.9 and 9.2, the majority of specimens (85.5%) had a RIN > 6.0. RINs were lower when specimens were collected from patients that were (i) hospitalized before death compared to those that were not (p<0.006), (ii)female rather than male (p<0.05) and (iii) diagnosed with Alzheimer’s disease rather than amyotrophic lateral sclerosis (ALS, p<0.0001), multiple sclerosis (MS, p<0.001) or no neurological conditions (p<0.0001). Further, RIN was weakly and negatively correlated with patient age (r=-0.252, p<0.001), modestly correlated with pH (r=0.598, p<0.05), and weakly and negatively correlated with agonal events (duration of hospitalization, coma duration, occurrence of respiratory illness, and duration of artificial ventilation) (r = 0.335; p< 0.01). Further, ANOVA showed the number of agonal events significantly affected RIN (p<0.01), and a significant difference in RIN among specimens collected from patients that did not experience an  agonal event compared to those that experienced 1 (7.1 versus 6.45, p<0.01) or 2 (7.1 versus 6.35 p<0.01). However, RIN was not significantly affected by brain region, brain weight, CSF pH, freezing method, coma duration, presence of respiratory illness, duration of ventilation, postmortem interval, duration of frozen storage, month of death, duration that the body was stored at room temperature or in cold storage, time between death and autopsy or autopsy and freezing, transport of the brain at ambient temperatures rather than chilled, or the duration of dissection. As expected, RIN was significantly correlated with mRNA levels of β-actin (r=0.724, p<0.0001), β2-microglobuiln (r=0.608, p<0.0001), beclin-1 (r=0.605, p<0.0001), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, r=0.629, p<0.0001), cyclophilin A (r=0.625, p<0.0001) and β-tubulin (r=0.5246, p=0.0003), but not 18S rRNA (r=-0.112 p=0.476). However, 18S rRNA was modestly correlated with PMI (r=-0.48, p=0.001), while RNA yield was not affected. The number of agonal events a patient experienced was modestly correlated with β-actin (r=-0.312, p=0.0416) and cyclophilin A (r=-0.417, p=0.0054) mRNA levels. HIF1α levels were also significantly higher in specimens from patients with one or more agonal events than patients that did not experience one (p<0.05). The ranking of most stable to least stable housekeeping genes was 18S rRNA, Beclin-1, β-actin, B2M, GAPDH, PP1a and β-tubulin.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Alzheimer's Disease
   • Amyotrophic Lateral Sclerosis
   • Other diagnoses
   • Autopsy
   • Parkinson's Disease
   • Multiple Sclerosis

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	0.5-101 h
   Preaquisition	Diagnosis/ patient condition	Hospitalized Not hospitalized 0-3 Agonal events Duration of artificial ventilation (1-3 days) Coma No coma Respiratory illness Alzheimer’s disease Amyotrophic lateral sclerosis Multiple sclerosis Parkinson disease Non-neurological condition
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	20-92 years
   Biospecimen Acquisition	Biospecimen location	Superior frontal gyrus gray matter Superior frontal gyrus white matter Cerebellum white matter Cerebellum gray matter Thalamus Corpus callosum
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB B2M BECN1 GAPDH HIF1A PP1A RRN18S TUBB
   Storage	Storage duration	Frozen for 0-120 months Refrigerated for 0-26 h
   Biospecimen Acquisition	Time of biospecimen collection	January February March April May June July August September October November December
   Preaquisition	Rapidity of death	0-3 Agonal events
   Biospecimen Acquisition	Organ measurements	850-1700 g
   Biospecimen Preservation	Cooling or freezing method/ rate	Liquid nitrogen Dry ice Pre-cooled isopentane, multiple cooling methods examined
   Biospecimen Aliquots and Components	pH	5 6 7 8 9
   Preaquisition	Postmortem condition(s)	Refrigerated storage Room temperature storage
   Storage	Specimen transport duration/condition	Cold Ambient

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