RNA stability in human liver: comparison of different processing times, temperatures and methods.
Author(s): Lee SM, Schelcher C, Gashi S, Schreiber S, Thasler RM, Jauch KW, Thasler WE
Publication: Mol Biotechnol, 2013, Vol. 53, Page 1-8
PubMed ID: 22271457 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA integrity and transcript stability in liver specimens procured by biopsy to surgically-resected specimens subjected to 3 or 24 h of cold ischemia at room temperature or on wet ice. Potential effects of preserving specimens by snap-freezing in liquid nitrogen or immersion in RNAlater prior to storage at -80°C was also examined.
Conclusion of Paper
RIN was modestly, albeit significantly, reduced in liver specimens subjected to cold ischemia times that approximated 24 h at room temperature compared to biopsy specimens procured prior to surgery or specimens subjected to delays on wet ice or at room temperature for shorter durations (p<0.05). Gene expression was stable when specimens were stored on wet ice for up to 24 h or at room temperature for 3 h or less. Further, significant declines in gene expression were limited to GUSB and HPRT1 and predominantly occurred in specimens stored at room temperature for 24 h after pathology processing (p<0.05). With regard to preservation method, RIN and HPRT1 and GUSB gene expression were significantly higher in biopsy specimens preserved in RNAlater compared to those snap-frozen in liquid nitrogen (p<0.05).
Studies
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Study Purpose
This study compared RNA integrity and transcript stability in liver specimens procured by biopsy to surgically-resected specimens subjected to cold ischemia at room temperature or on wet ice. Normal adjacent liver specimens were collected from six patients immediately prior to surgery by biopsy, immediately after surgery (designated 0 h), after processing by the pathology department, 3 h after processing by pathology, and 24 h after processing by pathology. Specific warm ischemia times and the amount of time that transpired between completion of surgery and processing by pathology were not provided. Liver specimens were preserved by snap-freezing in liquid nitrogen or immersion in RNAlater immediately or following cold ischemia, either at room temperature or on wet ice. All specimens were stored at -80°C after preservation. The stability of presumed reference genes HPRT1 and GUSB during ischemia was investigated as well as the impact on expression of FOS, GFER, HIF1a, and MYC following normalization to the reference gene HPRT1.
Summary of Findings:
RIN was modestly, albeit significantly, higher in liver specimens preserved with RNAlater compared to those snap-frozen in liquid nitrogen in biopsies collected prior to surgery (2% increase), as well as surgically-resected specimens preserved after processing by pathology (3.7% increase; p<0.05). Ischemia attributable differences in RIN were limited to a significant decline of approximately 20% in liver specimens stored at room temperature for 24 h after processing by pathology compared to biopsy specimens or those stored on wet ice or for shorter durations (p<0.05); the magnitude of RIN decline was similar for RNAlater and snap-frozen specimens. HPRT1 and GUSB gene expression (determined by the 2 delta CT method) was significantly lower in biopsies that were snap-frozen in liquid nitrogen compared to those preserved in RNAlater (p<0.05), as well as in liver specimens stored at room temperature for 24 h after processing by pathology compared to specimens stored on wet ice or for shorter durations regardless of preservation method (p<0.05). HPRT1 levels were also significantly higher in RNAlater-preserved specimens at the 3 h post-pathology time point compared to snap-frozen specimens (p<0.05). When gene expression was normalized to HPRT1, ischemia attributable differences were limited to a 22-26% decline in GUSB in RNAlater-preserved liver specimens stored at room temperature for 24 h after processing by pathology compared to biopsy specimens or specimens stored on wet ice or for shorter durations (p<0.05); conversely, normalized expression of FOS, GFER, HIF1a and MYC remained stable.
Biospecimens
Preservative Types
- RNAlater
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 h
3 h
24 h
Real-time qRT-PCR Specific Targeted nucleic acid FOS
GFER
GUSB
HIF1A
HPRT
MYC
Biospecimen Acquisition Method of tissue acquisition Surgical resection
Biopsy
Storage Storage temperature Room temperature
Wet ice