Differential long-term stability of microRNAs and RNU6B snRNA in 12-20 year old archived formalin-fixed paraffin-embedded specimens.
Author(s): Peskoe SB, Barber JR, Zheng Q, Meeker AK, De Marzo AM, Platz EA, Lupold SE
Publication: BMC Cancer, 2017, Vol. 17, Page 32
PubMed ID: 28061773 PubMed Review Paper? No
Purpose of Paper
This paper investigated the potential effects of formalin-fixed, paraffin-embedded (FFPE) prostate tissue block storage on RNA integrity and levels of miRNA and snRNA as determined by real-time RT-PCR.
Conclusion of Paper
There was a significant inverse correlation between FFPE block storage duration (12-20 y) and transcript level for each of the four non-coding RNAs evaluated (RNU6B, rho= -0.685; miR-21, rho= -0.527; miR-221, rho= -0.493; miR-141, rho= -0.314; P<0.001 for all). A significant positive correlation between RNA Integrity Number (RIN) and real-time RT-PCR levels was limited to miR-141 levels (P<0.001), although RINs were notably low for all FFPE specimens (RIN range=1-3.9). When transcript-specific stability was examined using general linear models, results revealed that miR-21 and RNU6B had comparable and faster degradation rates than miR-141 and miR-221, which were significantly more stable (P<0.001).
Studies
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Study Purpose
This study investigated potential effects of formalin-fixed, paraffin-embedded (FFPE) prostate tissue block storage on RNA integrity and levels of miRNA and snRNA as determined by real-time RT-PCR. Two 0.6-mm cores containing tumor tissue were obtained from 92 radical prostatectomy specimens that had been formalin-fixed and paraffin-embedded and stored as bocks for 12 to 20 y at a single institution using standardized methods. Details of formalin fixation and paraffin processing were not provided. Cores were deparaffinized in xylene prior to RNA extraction with Ambion's RecoverAll Total Nucleic Acid Isolation kit. RNA quantity was determined by Nanodrop spectrophotometry and integrity by RIN with a bioanalyzer. Levels of miRNA (miR-21, miR-141, miR-221, and RNU6B) were determined by Taqman real-time RT-PCR analysis and standard curves for each transcript of interest were generated using cell lines. Spearman rank correlations were used to identify significant correlations between RIN and FFPE block storage duration.
Summary of Findings:
There was a significant inverse correlation between FFPE block storage duration and transcript quantity for each of the four non-coding RNAs evaluated in blocks that were stored between 12 and 20 y prior to RNA extraction and analysis (P<0.001 for all). The strength of the correlations ranged from modest (RNU6B, rho= -0.685; miR-21, rho= -0.527; miR-221, rho= -0.493) to weak (miR-141, rho= -0.314). A significant positive correlation between RIN and real-time RT-PCR levels was limited to miR-141 levels (P<0.001), although RINs were notably low for all FFPE specimens (RIN range=1-3.9). When transcript-specific stability was examined using general linear models, results revealed that miR-21 and RNU6B had comparable and faster degradation rates than miR-141 and miR-221, which were significantly more stable (P<0.001).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Targeted nucleic acid miR-21
miR-141
miR-221
RNU6B
Storage Storage duration 12-20 y