RNA integrity in post mortem human variant Creutzfeldt-Jakob disease (vCJD) and control brain tissue.

Author(s): Sherwood KR, Head MW, Walker R, Smith C, Ironside JW, Fazakerley JK

Publication: Neuropathol Appl Neurobiol, 2011, Vol. 37, Page 633-42

PubMed ID: 21251044 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if RNA yield and integrity are influenced by patient diagnosis, preservation method, recurrent sampling of frozen specimens, or the duration of storage at -80 degrees C. Specimens were collected from the second gyris from the midline at autopsy or from archived brain specimens frozen as intact halves at -80 degrees C. Specimens collected at autopsy were snap frozen in liquid nitrogen or immersed in RNAlater prior to storage at -80 degrees C. Brain specimens from 78 individuals were analyzed. Since sampling of intact frozen brain halves required partial thawing, the potential effect of multiple sampling events on RIN was also examined.

   Summary of Findings:

   Brain specimens preserved in RNAlater at autopsy had significantly higher RINs compared to specimens that were snap frozen in liquid nitrogen or at -80 degrees C as intact brain halves (p<0.05), although RINs of frozen specimens did not differ from one another. The RNA integrity of frozen specimens was poor, as the majority of specimens had a RIN below 5. No significant differences in RIN were observed between disease states for any of the preservation methods examined. While the authors report that RNA yield and RIN were not correlated, data was not shown. The number of sampling events, which required partial thawing of intact frozen brain specimens, had a significant and detrimental impact on RIN (p<0.0001). Identification of a threshold of effect was not possible due to high variability among brains subjected to a low number of sampling events. RINs were not significantly affected by the duration of frozen storage in a different sample set of 20 specimens snap frozen in liquid nitrogen; therefore, the authors conclude that the decline in RIN among frozen brain halves is attributable to thawing. Differences in RNA yields were not significantly affected by the number of sampling events or the duration of frozen storage, although variability was high among both specimen groups.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Autopsy
   • Lewy Body Disease
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen RNAlater Snap frozen
   Storage	Storage duration	2-17 months
   Storage	Freeze/thaw cycling	1 cycle 25 cycles
   Preaquisition	Diagnosis/ patient condition	Creutzfeldt-Jakob disease Lewy Body disease Cerebrovascular disease Atherosclerosis Unspecified non-neurological diseases

   View more

2. Study Purpose

   The purpose of this study was to determine if RNA yield and integrity of brain specimens are influenced by age at time of death, diagnosis, gender, PMI, or brain pH. A total of 53 specimens collected from the second gyris from the midline and snap frozen at autopsy or from archived frozen brain specimens sampled less than five times were analyzed. All specimens were stored at -80 degrees C until analysis. Study Findings

   Summary of Findings:

   RIN was not significantly nor strongly correlated to age at time of death, diagnosis, PMI, or brain pH for either gender. Further, although a significant negative correlation was observed between RNA yield and age at death (p<0.05) the strength of the correlation was very weak (r2=0.007), and no significant correlations were observed between RNA yield and diagnosis, PMI, or brain pH for either gender.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Autopsy
   • Lewy Body Disease
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	16-86 y
   Preaquisition	Patient gender	Female Male
   Preaquisition	Postmortem interval	6-288 h
   Biospecimen Aliquots and Components	pH	5.6 6.7
   Preaquisition	Diagnosis/ patient condition	Creutzfeldt-Jakob disease Lewy Body disease Cerebrovascular disease Atherosclerosis Unspecified non-neurological diseases
   Biospecimen Preservation	Cooling or freezing method/ rate	At -80 degrees C Liquid nitrogen

   View more