Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity.
Author(s): Yi QQ, Yang R, Shi JF, Zeng NY, Liang DY, Sha S, Chang Q
Publication: J Int Med Res, 2020, Vol. 48, Page 300060520931259
PubMed ID: 32567435 PubMed Review Paper? No
Purpose of Paper
This paper investigated the potential effects of formalin-fixed, paraffin-embedded (FFPE) block storage for 1-8 y at room temperature (25°C) on DNA and RNA concentrations, purity, integrity and amplificability of the β-globulin and aldehyde dehydrogenase-2 (ALDH2) genes.
Conclusion of Paper
Compared to FFPE blocks stored in the dark at 25°C for 1-3 y, those stored for 8 y had significantly lower DNA and RNA concentrations (p<0.05 for all) and more severe DNA and RNA degradation. However, no storage duration-related differences in purity (OD 260/280) or qPCR or qRT-PCR amplificability (Ct values and visual assessment of amplicons by gel electrophoresis) of β-globulin (268 bp) and ALDH2 (135 bp) genes and transcripts were observed. The authors concluded that FFPE blocks stored for up to 8 y in a light- and temperature-controlled environment are suitable for analysis of small DNA and RNA fragments after validation of target stability.
Studies
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Study Purpose
The purpose of this study was to determine if FFPE block storage for 1, 3, or 8 y in the dark at room temperature adversely affects DNA and RNA concentration, purity, integrity, and amplificability of two reference genes (β-globulin and ALDH2. Uterine fibroid specimens were collected from 20 patients diagnosed with hysteromyoma in 2010 (8 y of storage), 2015 (3 y of storage), or 2017 (1 y of storage)(60 patients in total). Specimens were fixed in formalin for 12 h prior to paraffin embedding (with paraffin with a melting temperature of 56°C) of a 1 x 1x 0.2 cm tumor segment. FFPE blocks were stored in the dark at 25°C for 1 y (specimens collected in 2017), 3 y (specimens collected in 2015), or 8 y (specimens collected in 2010). No other details on preservation and processing were reported. Six 10 µm-thick sections underwent deparaffinization in xylene (55°C for 10 min) and proteinase K digestion (DNA: 56°C for 1 h; RNA: 56°C for 15 min) before DNA and RNA extraction with the AllPrep DNA/RNA FFPE Kit. DNA and RNA concentration and purity were determined by spectrophotometry, and integrity was assessed by gel electrophoresis. Amplificability of DNA and RNA extracted from FFPE specimens stored as blocks for different durations were assessed by qPCR and qRT-PCR, respectively, of β-globulin (268 bp amplicon) and ALDH2 (135 bp) genes and transcripts.
Summary of Findings:
FFPE blocks displayed a significant decline in both DNA and RNA concentration with storage duration (1-3 y versus 8 y; p<0.05 for all), but no storage-related differences in purity (OD 260/280) were observed. While evidence of DNA and RNA degradation were observed among all FFPE blocks, blocks stored for 8 y displayed more prominent degradation than those stored for 1-3 y. Ct values of qPCR and qRT-PCR analyses of β-globulin and ALDH2 genes and transcripts and transcripts did not differ significantly among FFPE blocks store for 1-8 y; visualization of amplicons via gel electrophoresis also did not reveal any appreciable difference between specimens stored for different durations. The authors concluded that FFPE blocks stored for up to 8 y in a light- and temperature-controlled environment are suitable for analysis of small DNA and RNA fragments after validation of target stability.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis RNA Real-time qRT-PCR RNA Spectrophotometry RNA Electrophoresis DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 y
3 y
8 y
Real-time qRT-PCR Specific Targeted nucleic acid ALDH2
β-globulin
Real-time qPCR Specific Targeted nucleic acid β-globulin
ALDH2