RNA and microRNA Stability in PAXgene-Fixed Paraffin-Embedded Tissue Blocks After Seven Years' Storage.
Author(s): Sanchez I, Betsou F, Culot B, Frasquilho S, McKay SC, Pericleous S, Smith C, Thomas G, Mathieson W
Publication: Am J Clin Pathol, 2018, Vol. 149, Page 536-547
PubMed ID: 29659661 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of room temperature storage of PAXgene-fixed paraffin-embedded (PFPE) tissue blocks for 7 years on RNA and miRNA integrity.
Conclusion of Paper
Mean RNA integrity numbers (RINs) were comparable between RNA analyzed immediately after extraction and after storage at -80°C for 7 years, but median RIN and median normalized paraffin-embedded RNA metric (PERM) were significantly lower when RNA was extracted after PFPE tissue block storage at room temperature for 7 years. Amplicons ≥584 bp were obtained from 78% of specimens when RNA was extracted immediately after PAXgene fixation and paraffin embedding but were observed in only 6% of specimens when RNA was extracted after 7 years of storage. Similarly, cycle threshold (Ct) values were higher for β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and hydroxymethylbilane synthase (HMBS) in RNA from PFPE specimens stored for seven years compared to RNA extracted from blocks immediately after PAXgene fixation and paraffin embedding. Relative expression analysis of real-time qRT-PCR results revealed that while GAPDH and HMBS degraded at comparable rates with storage, β-actin was not affected to the same extent as either GAPDH or HMBS. Similar to RNA, all three miRNAs had mean Ct numbers that were higher in RNA from blocks stored for 7 years compared to RNA extracted immediately after PAXgene fixation and paraffin embedding, but the differences were not significant. Tissue morphology was unaffected by block storage and the authors state that differences observed in extent of eosinophilia was due to the use of different scanning systems and software.
Studies
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Study Purpose
This study investigated the effects of room temperature storage of PAXgene-fixed paraffin-embedded (PFPE) tissue blocks for 7 years and -80°C storage of extracted RNA on integrity of RNA and miRNA. Twenty-five tissue specimens (11 breast tumor, 5 liver, 3 prostate, 1 normal renal, 1 pelvic mass tumor, 1 normal colon, 1 ovary tumor, 1 myometrium tumor, 1 adrenal gland) were fixed using the PAXgene system and then embedded in paraffin. RNA and miRNA were extracted immediately from deparaffinized 20 μm sections using the PAXgene Tissue RNA kit and PAXgene Tissue miRNA kits, respectively, and stored at -80°C. The PFPE blocks were stored in the dark at room temperature for 7 years and RNA and miRNA were extracted from five 10 μm sections of the stored blocks. RNA and miRNA yields were determined spectrophotometrically and RINs assigned by a bioanalyzer. Paraffin-embedded RNA metric (PERM) values were calculated using the number of fluorescence units in the electropherogram at nine specified time points during the run. RT-PCR was performed using 65, 256, 584, and 942 bp amplicons of the hydroxymethylbilane synthase (HMBS) gene and visualized on an agarose gel. miRNA integrity was evaluated by real-time RT-PCR analysis of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and HMBS. Slides were prepared from 4 μm-thick sections and H&E stained for morphological assessment.
Summary of Findings:
Mean RINs were comparable between RNA analyzed immediately after extraction and after storage at -80°C for 7 years (3.59 and 3.63, respectively; P= 0.83), but median RIN and median normalized PERM were significantly lower when RNA was extracted after block storage at room temperature for 7 years (P<0.001 and P=0.008, respectively). Amplicons ≥584 bp were obtained from 78% of specimens when RNA was extracted immediately after PAXgene fixation and paraffin embedding but were observed in only 6% of specimens when RNA was extracted after 7 years of storage. Similarly, Ct values were higher for β-actin, GAPDH, and HMBS in RNA from PFPE specimens stored for seven years compared to RNA extracted from blocks immediately after PAXgene fixation and paraffin embedding (P=0.04, P<0.001, and P=0.002; respectively). Relative expression analysis of real-time qRT-PCR results revealed that while GAPDH and HMBS degraded at comparable rates with storage (mean ΔCt= 6.4 and 6.3, respectively; P=0.44), β-actin was not affected to the same extent as either GAPDH (mean ΔCt 1.7 versus 3.9, P<0.001) or HMBS (mean ΔCt 9.4 versus 8.2, P<0.001). Similar to RNA, mean Ct numbers were higher for all three miRNAs analyzed in RNA from blocks stored for 7 years compared to RNA extracted immediately (29.5 versus 27.2 for RNU24, 25.0 versus 24.9 for miR-16, and 29.3 versus 28.8 for miR-221), but the differences were not significant (P>0.09). Tissue morphology was unaffected by block storage and the authors state that differences observed in extent of eosinophilia was due to the use of different scanning systems and software.
Biospecimens
- Tissue - Breast
- Tissue - Liver
- Tissue - Prostate
- Tissue - Uterus
- Tissue - Colorectal
- Tissue - Ovary
- Tissue - Adrenal Gland
- Tissue - Kidney
Preservative Types
- PAXgene
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR Morphology H-and-E microscopy RNA Spectrophotometry RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 years
7 years
RT-PCR Specific Targeted nucleic acid hydroxymethylbilane synthase (HMBS)
Real-time qRT-PCR Specific Targeted nucleic acid miR-16
miR-221
RNU24
Storage Storage conditions Extracted RNA
PFPE block