Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies.
Author(s): Carlsson J, Davidsson S, Fridfeldt J, Giunchi F, Fiano V, Grasso C, Zelic R, Richiardi L, Andrén O, Pettersson A, Fiorentino M, Akre O
Publication: BMC Med Res Methodol, 2018, Vol. 18, Page 161
PubMed ID: 30518332 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effect of extraction kit choice by comparing the yield, purity, and integrity of RNA and DNA isolated from matched serial sections of archival histologically normal prostate biopsies following extraction with different kits designed for formalin-fixed paraffin-embedded (FFPE) specimens. The authors also investigated how tumor length, percentage tumor cells, Gleason scores, and block storage duration affected the yield, purity, integrity, and CT values of the RNA and DNA extracted from FFPE sections of prostate cancer biopsies.
Conclusion of Paper
The RNeasy FFPE Kit yielded RNA with a higher median RNA integrity number (RIN) than the HighPure Kit, FFPE RNA Micro Kit, or the AllPrep DNA/RNA FFPE Kit. The RNeasy Kit also yielded more RNA and the RNA obtained had a higher A260/280 than that obtained using the HighPure Kit. Use of the QIAamp Kit resulted in DNA of much higher mean purity than either the HighPure or AllPrep kits but the mean cycle threshold (CT) values for 18s rRNA were slightly higher for DNA extracted using the QIAamp Kit than the other kits.
In specimens from prostate cancer patients, significant correlations were identified between tumor length and RNA/DNA yield and RIN values and significant differences in RNA yield, DNA yields, and RNA CT values for 18s rRNA were found when specimens were classified by tumor length. The authors state the percentage of tumor cells and Gleason scores were not correlated with DNA or RNA yield, A260/A280 ratios, RIN, or the DNA or RNA CT-values for 18s rRNA. However, there was a weak correlation between age of the FFPE tissue and DNA yield but not RNA yield. DNA and RNA yield from matched sections were not correlated. For RNA, there were significant negative weak to modest correlations identified between yield and A260/A280 and between the CT values for 18s rRNA and between RIN and A260/A280. For DNA, a weak negative correlation was observed between the yield and the CT value for 18s rRNA.
Studies
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Study Purpose
This study compared three RNA extraction kits designed for FFPE specimens by comparing the yield, purity, and integrity of RNA isolated from matched serial sections of archival histologically normal prostate biopsies. Twenty FFPE prostate needle biopsies with normal histology collected between 1992 and 2002 were obtained from the archive (no further details of processing were provided). RNA was extracted from freshly-cut serial 10 µm sections of ten biopsies using the High Pure FFPE RNA Micro Kit and the RNeasy FFPE Kit. RNA was extracted from 10 µm sections of the remaining ten biopsies using the AllPrep DNA/RNA FFPE Kit and the RNeasy FFPE Kit and purity and yield were measured using a spectrophotometer. RNA integrity was assessed using a bioanalyzer and by TaqMan real-time RT-PCR of the 18S rRNA transcript.
Summary of Findings:
In the comparison between the HighPure FFPE RNA Micro Kit and the RNeasy FFPE Kit, the RNeasy Kit yielded RNA with significantly higher mean yield (16.3 ng/µL versus 5.6 ng/µL, P<0.001), purity (A260/280 of 1.55 versus 1.38, P=0.003), and median RIN (2.15 versus 1.0, P=0.011) but a comparable number of amplifiable copies of 18S rRNA transcript (mean cycle threshold of 25.4 versus 26.4, P=0.268) to the HighPure Kit. Importantly, Bland-Altman plots indicated that the higher yield, purity, and RIN with RNeasy were fixed bias and there was also a proportional bias in RIN (slope: 1.98, P<0.0001). In the comparison between the RNeasy FFPE Kit and the AllPrep DNA/RNA FFPE Kit, the RNeasy Kit produced RNA with a higher median RIN value (2.5 versus 1.0, P=0.044) but there was no difference in mean yield (11.4 ng/µL versus 12.6 ng/µL, P=0.307), purity (A260/280 of 1.47 versus 1.50, P=0.419), or amplifiable copies of 18S rRNA transcript (mean cycle threshold of 27.4 versus 28.0, P=0.088). Bland-Altman plots confirmed a fixed bias toward higher RIN with RNeasy, but in this comparison, the bias was not found to also be proportional.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method High Pure FFPE RNA Micro kit
RNeasy FFPE kit
AllPrep DNA/RNA FFPE kit
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Study Purpose
This study compared three DNA extraction kits designed for FFPE specimens by comparing the yield, purity, and integrity of DNA isolated from matched serial sections of archival histologically normal prostate biopsies. Twenty FFPE prostate needle biopsies with normal histology collected between 1992 and 2002 were obtained from the archive (no further details of processing were provided). DNA was extracted from freshly-cut serial 10 µm sections of ten biopsies using the High Pure FFPET DNA Isolation Kit and QIAamp DNA FFPE Tissue Kit. DNA was extracted from 10 µm sections of the remaining ten biopsies using the AllPrep DNA/RNA FFPE Kit and the QIAamp DNA FFPE Tissue Kit. DNA purity and yield were measured using a spectrophotometer. Integrity of 18S rRNA DNA was evaluated by TaqMan real-time PCR of the 18S rRNA transcript.
Summary of Findings:
DNA yields were comparable between kits in both comparisons with no extraction method-based bias in yield identified. Use of the QIAamp Kit resulted in DNA of much higher mean purity than either the HighPure (A260/A230 of 1.89 versus 1.37, P=0.002) or AllPrep (A260/A230 of 1.68 versus 1.36, P<0.001) kits and, in both cases, a fixed bias toward higher purity in specimens obtained with QIAamp was identified. However, the mean cycle threshold values for 18s rRNA were slightly higher for DNA extracted using the QIAamp Kit than that obtained using the HighPure (28.6 versus 27.1, P=0.011) or AllPrep (29.0 versus 27.3, P<0.001) kits.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method High Pure DNA FFPET Isolation kit
QIAamp DNA FFPE kit
AllPrep DNA FFPE kit
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Study Purpose
This study investigated how tumor length, percentage tumor cells, Gleason scores, and block storage duration affected the yield, purity, integrity, and CT values of the RNA and DNA extracted from FFPE sections of prostate cancer biopsies. Additionally, the correlation among yield, purity, and CT values were also examined. FFPE prostate needle biopsies from 84 patients with prostate cancer collected between 1992 and 2002 were obtained from the archive (no further details of processing were provided). Two pathologists examined the biopsies for percentage tumor (5-90%) and Gleason score and to identify tumor area. The tumor area was macrodissected from two 10 µm sections. RNA was extracted from one section using the RNeasy FFPE Kit and DNA was extracted from the other section using the QIAamp DNA FFPE Tissue Kit. DNA and RNA purity and yield were measured using a spectrophotometer. RNA integrity was assessed using a bioanalyzer. Integrity of 18S rRNA DNA/RNA was evaluated by TaqMan real-time PCR/RT-PCR of the 18S rRNA transcript. The significance of differences was evaluated using Student’s t-test and a Wilcoxon matched pairs signed rank tests and correlations investigated using the Pearson correlation coefficient.
Summary of Findings:
DNA and RNA were obtained from all sections but one RNA extract and three DNA extracts did not allow for amplification of the 18S rRNA transcript at a CT<38. Significant but weak to modest correlations were identified between tumor length and RNA yield (r=0.45, P<0.001), DNA yield (r=0.45, P<0.001) and RIN values (r=0.37, P<0.01). Compared to specimens with tumor length of <5 mm, specimens ≥10 mm tumor length had higher RNA and DNA yields(P<0.001 and P<0.01, respectively) and lower RNA CT values for 18s rRNA (P<0.05) and specimens with 5-9 mm tumor length had higher DNA yields (P<0.01) and lower RNA CT values for 18s rRNA (P<0.05). The authors state that no correlations were found between percentage of tumor cells or Gleason scores with DNA or RNA yield, A260/A280 ratios, RIN, or the DNA or RNA CT-values for 18s rRNA. However, there was a weak correlation between age of the FFPE tissue and DNA yield (r=0.337, P<0.01), but not RNA yield. DNA and RNA yield from matched sections were not found to be correlated. For RNA, there were significant negative weak to modest correlations identified between yield and A260/A280 (r=-0.23, P<0.05) and between the CT values for 18s rRNA and RIN (r=-0.29, P<0.05) and A260/A280 (r=-0.51 P<0.001). For DNA, a weak negative correlation was observed between the yield and the CT value for 18s rRNA (r=-0.31, P<0.05).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Real-time qPCR RNA Real-time qRT-PCR RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Prognostic factor Gleason score ≤ 6
Gleason score 3+4
Gleason score 4+3
Gleason score 8-10
0.2-17.8 mm tumor
<5 mm tumor
5-9 mm tumor
≥10 mm tumor
Storage Storage duration Stored from 1992-2002 to study
Biospecimen Aliquots and Components Biospecimen heterogeneity A range of tumor cell contents examined