Effects of fixation and storage of human tissue samples on nucleic acid preservation.
Author(s): Nam SK, Im J, Kwak Y, Han N, Nam KH, Seo AN, Lee HS
Publication: Korean J Pathol, 2014, Vol. 48, Page 36-42
PubMed ID: 24627693 PubMed Review Paper? No
Purpose of Paper
This paper compared PCR-based amplification of DNA and RNA isolated from formalin-fixed, paraffin-embedded (FFPE) and methacarn-fixed, paraffin-embedded (MFPE) specimens. Durations of fixation, decalcification, ethanol storage and block storage was also investigated using FFPE specimens.
Conclusion of Paper
RNA isolated from FFPE specimens had higher yield (637 vs. 149 ng/uL, p<0.001) and purity (1.97 vs. 1.85, p<0.001) compared to MFPE specimens, although real-time RT-PCR results were comparable among FFPE and MFPE specimens. DNA isolated from FFPE and MFPE successfully generated amplicons 148 bp in length or shorter for all specimens; however, PCR success rate (100 vs. 47%, p-0.002) and the yield of a 434 bp amplicon were greater among MFPE than FFPE specimens. When time in formalin was investigated, DNA isolated from specimens fixed for 3 days or longer failed to generate a 434 bp amplicon and had reduced yields for shorter amplicons (60 and 148 bp). While RNA yield was higher among specimens fixed in formalin for 90 and 180 days compared to those fixed for 3-30 days (540 vs. 286 ng/uL, p=0.026), RNA purity and generation of a beta-actin real-time RT-PCR amplicon did not differ as a result of time in formalin. Storage of formalin-fixed specimens in 70% ethanol for 2 to 17 days at 4°C did not affect PCR success rate or amplicon yield. The duration of decalcification adversely affected RNA yield and purity, and real-time RT-PCR success rates in a time-dependent manner. PCR amplicon yields also significantly declined with longer decalcification times, although the magnitude of decline was also influenced by amplicon length. The duration of FFPE block storage (<1-8 y) did not alter DNA yield, quality, of PCR success rate; however RNA yield was lower among specimens stored for less than 1 month compared to those stored for more than 1 month (637 vs. 499 ng/uL; p=0.037) while RNA purity was lower among specimens stored for 3-8 years compared to those stored for less than 1 year (1.88 vs. 1.97, p=0.008). FFPE block storage also increased Ct values for beta-actin, with significant differences reported for blocks stored for less than 1 month (33.73), between 1 month and 1 year (35.19), and for 3-8 years (37.83; p=0.003).
Studies
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Study Purpose
This study compared DNA and RNA PCR-based amplification among specimens fixed in formalin or methacarn and then embedded in paraffin. Gastric and colorectal specimens were preserved by fixation in 10% neutral buffered formalin for 24-48 h or methacarn solution (60% methanol, 30% chloroform, 10% acetic acid) for an unspecified duration. Six 8 µm-thick sections were used for DNA extraction using Bio-Rad's InstaGene Matrix chelating ion-exchange resin , while eleven 8 µm-thick sections were used for RNA extraction with the High Pure RNA Paraffin kit. GAPDH (60 bp), beta-actin (148 bp) and hGH (434 bp) PCR amplicons were then generated using extracted DNA from 15 FFPE and 15 MFPE specimens, while beta-actin (97 bp) real-time RT-PCR amplicons were generated using extracted RNA from 12 FFPE and 12 MFPE specimens. RNA yield and purity (260/280 OD ratio) were determined by spectrophotometer.
Summary of Findings:
Generation of PCR amplicons ≤ 148 bp in length were successful for DNA isolated from all FFPE and MFPE specimens, while MFPE specimens had a higher PCR success rate than FFPE specimens for the 434 bp hGH amplicon (100 vs. 47%, p=0.002). Amplicon yield was also greater for DNA isolated from MFPE compared to FFPE specimens for beta-actin (148 bp) and hGH (434 bp). Conversely, higher RNA yield (637 vs. 149 ng/uL, p<0.001) and purity (1.97 vs. 1.85, p<0.001) were obtained from FFPE specimens compared to MFPE specimens, but real-time RT-PCR was successful for all FFPE and MFPE specimens and cycle threshold (Ct) values for beta-actin did not differ significantly between FFPE and MFPE specimens.
Biospecimens
Preservative Types
- Other Preservative
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Methacarn
PCR Specific Length of gene fragment 60 bp
148 bp
434 bp
Real-time qRT-PCR Specific Targeted nucleic acid beta-actin
PCR Specific Targeted nucleic acid GAPDH
beta-actin
hGH
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Study Purpose
This study evaluated impacts of the duration of fixation, decalcification, and ethanol storage on the amplification of DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) specimens. Five gastrointestinal surgical resected specimens from cancer patients were used to evaluate the impact of time in formalin, storage of formalin-fixed specimens in 70% ethanol at 4°C, and the duration of decalcification on PCR amplification of DNA and real-time RT-PCR amplification of RNA. Six 8 µm-thick sections were used for DNA extraction using Bio-Rad's InstaGene Matrix chelating ion-exchange resin, while eleven 8 µm-thick sections were used for RNA extraction with the High Pure RNA Paraffin kit. Extracted DNA was analyzed by PCR for the following amplicons: GAPDH (60 bp), beta-actin (148 bp) and hGH (434 bp). Extracted RNA was analyzed by real-time RT-PCR for beta-actin (97 bp). RNA yield and purity (260/280 OD ratio) were determined by spectrophotometer. The impact of FFPE block storage on DNA yield and quality, RNA yield and purity, and DNA and RNA PCR and real time RT-PCR success, respectively, was also investigated in 30 FFPE blocks of unspecified tissue origin that were stored for less than a month up to 8 years.
Summary of Findings:
DNA isolated from specimens fixed in formalin fixation for 3 days or longer failed to generate the 434 bp amplicon by PCR, and adversely affected yields of 60 and 148 bp PCR amplicons. While RNA yield was greater among specimens fixed in formalin for 90 and 180 days compared to those fixed for 3-30 days (540 vs. 286 ng/uL, p=0.026), RNA purity and generation of a beta-actin real-time RT-PCR amplicon were not significantly affected by time in formalin. While effects on RNA yield and real-time RT-PCR analysis of beta-actin were not reported, storage of formalin-fixed specimens in 70% ethanol for 2 to 17 days at 4°C did not affect PCR success rates or amplicon yield.
The duration of decalcification adversely affected PCR and real-time RT-PCR success rates, and RNA yield and purity. When DNA was analyzed by PCR, the number of specimens that successfully generated an amplicon declined as decalcification time progressed for amplicons of all sizes (GAPDH, 60 bp, p=0.017; beta-actin, 148 bp, p<0.001; hGH, 434 bp, p<0.001), but the magnitude of decline was also proportional to the length of the amplicon. RNA yield and purity exhibited linear decreases with longer decalcification periods (p=0.004 and p=0.009, respectively) and real-time RT-PCR success rates for beta-actin (97 bp) declined significantly as decalcification time increased (10 min: 80%, 30 min: 0%, 60 min: 40%, 120 min: 20%, 180 min: 20%; p=0.009).
DNA yield, quality, or PCR success rates did not differ among FFPE specimens stored as paraffin blocks for less than 1 month, for 6 months to 1 year, or for up to 8 years. However, RNA yield was significantly higher among specimens stored for 6 months to 1 year compared to controls stored for less than 1 month (499 vs. 637 ng/uL; p=0.037), while RNA purity was significantly lower among those stored for 3-8 years compared to those stored for 6 months to 1 year (1.88 vs. 1.97, p=0.008). FFPE block storage also increased real-time RT-PCR Ct values, with significant differences reported for blocks stored for less than 1 month (33.73), between 6 months and 1 year (35.19), and for 3-8 years (37.83; p=0.003).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 3 d
7 d
30 d
90 d
180 d
PCR Specific Length of gene fragment 60 bp
148 bp
434 bp
Real-time qRT-PCR Specific Targeted nucleic acid beta-actin
PCR Specific Targeted nucleic acid GAPDH
beta-actin
hGH
Storage Storage duration < 1 month
6 months
1 y
3 y
5 y
8 y
Analyte Extraction and Purification Decalcification solution/ duration 0 min
10 min
30 min
60 min
120 min
180 min
Storage Storage in ethanol (duration/condition) 2 d
6 d
29 d
179 d