Prolonged Room Temperature Ischemia Does Not Affect the Quality of Total Tissue RNA
Larry True, Belinda Nghiem, Bryce Lakely, Colm Morrissey. University of Washington, Seattle
Background: Molecular assays provide guidance for identifying tumors likely to respond to targeted therapy. Many of these assays require high quality RNA. A method widely used to assess RNA quality uses the Bioanalyzer (Agilent). Degree of RNA integrity is reported as RNA Integrity Numbers (RIN), using a Bioanalyzer. RINs range from 1 to 10 (1 indicates degradation of total RNA; 10 indicates little or no RNA degradation). We hypothesized that the length of time a samples sits at room temperature before freezing (the cold ischemia time) correlates with extent of RNA degradation.
Design: Prostate cancer and benign tissues were acquired from both rapid autopsy (RA; n=35) and radical prostatectomy (RP; n=72) samples. Cold ischemia times were recorded. Integrity of total RNA extracted from tissue aliquots was determined on an Agilent 2100 Bioanalyzer.
Results: RINs of RP samples (range of cold ischemia time: 20 to 100 min.) ranged from 2.4 to 9.7. There was no correlation of RIN with time.
RINs of RA samples (range of time from patient death to tissue stabilization by freezing: 2 to 6.5 hrs.) ranged from 3.0 to 7.9 (Different samples from each autopsy differed in RIN).
With several exceptions, there was a trend toward decreasing RIN with time, though some samples after 5 hours of cold ischemia still had a RIN of 7.
Conclusions: Despite prolonged cold ischemia times (up to 2 hrs for RP samples and 6.5 hrs. for RA samples), the quality of RNA does not significantly decrease in a time-dependent manner. Pre-analytical factors, i.e. warm ischemia time, hypoxia, high protein pre-op diet, etc., are known to affect levels of specific mRNA's. Since the quality of total RNA was essentially independent of cold ischemia time, we cannot rely upon the RIN to reflect the effect of cold ischemia time on RNA quality for tissue acquired within 7 hours of devitalization. To predict which tissue samples have high quality RNA, a better assay of RNA integrity is needed.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 279, Tuesday Morning