Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.
Author(s): Einaga N, Yoshida A, Noda H, Suemitsu M, Nakayama Y, Sakurada A, Kawaji Y, Yamaguchi H, Sasaki Y, Tokino T, Esumi M
Publication: PLoS One, 2017, Vol. 12, Page e0176280
PubMed ID: 28498833 PubMed Review Paper? No
Purpose of Paper
This paper investigated how total and amplifiable yields of DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue may be affected by the duration, concentration, and temperature of formalin fixation, as well as inclusion of a heat-treatment step prior to extraction. Step-by-step analysis of the potential impact immunohistochemistry (IHC) staining may have on the yield of amplifiable and total DNA from FFPE specimens was also conducted. Discrepancies in next generation sequencing (NGS) data obtained using case-matched FFPE and frozen normal liver specimens were further examined by Sanger sequencing or Real-time PCR.
Conclusion of Paper
The yield of total DNA isolated from FFPE specimens did not appear to be adversely affected by the concentration of formalin (20% versus 10%), fixation duration (2-9 d), or the temperature of fixation (25˚C versus above 25˚C ), but total yield decreased when specimens were subjected to antigen retrieval for immunohistochemical staining. In contrast, the amount of amplifiable DNA was lower in FFPE specimens than frozen specimens and decreased further when specimens were fixed in 20% formalin (as opposed to 10% formalin), fixed (in 10% formalin) for more than 3 days, or fixed at temperatures above 25˚C. Incubating samples obtained from FFPE specimens at 95˚C for 30 min following protease digestion resulted in an increase in amplifiable DNA. Specimens that were subjected to antigen retrieval before DNA extraction had much less amplifiable DNA.
Next generation sequencing of FFPE specimens identified 19 mutations not found in case-matched frozen specimens. However, further investigation by Sanger sequencing and allele-specific real-time PCR was not able to confirm three and six of the mutations, respectively. The authors concluded that the unconfirmed mutations identified by NGS were more likely an artifact of NGS than of the formalin fixation process.
Studies
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Study Purpose
This study investigated how total and amplifiable yields of DNA isolated from FFPE specimens may be affected by formalin fixation, formalin concentration, and the duration and temperature of fixation, as well as inclusion of a heat-treatment step prior to extraction. Specimens included 10 normal liver, 4 normal breast specimens, 7 liver tumors (metastasis of colorectal cancer and hepatocellular carcinomas) and 6 tongue tumors. One liver, 2 breast and 5 tongue specimens were fixed in 10% formalin at ≤25˚C for 2 days; two liver specimens were fixed in 10% formalin at >25˚C for 2 days; four liver specimens were fixed in 10% formalin at ≤25˚C for 3 days, one liver specimen was fixed in 10% formalin at >25˚C for 3 days; two liver and one tongue specimen were fixed in 10% formalin at ≤25˚C for 4 days; one liver specimen was fixed in 10% formalin at >25˚C for 4 days; two breast specimens were fixed in 20% formalin at ≤25˚C for 2 days; and, the remaining 5 liver specimens were fixed in 20% formalin at ≤25˚C for 3, 5, 5, 5 and 9 days, respectively. Tissue specimens were then processed and embedded in paraffin, but details were not provided. Additionally, case-matched frozen specimens were also obtained from ten normal livers and stored at -80˚C. After overnight protease digestion DNA was extracted from frozen specimens using phenol-chloroform and from FFPE specimens using the RecoverAll Total Nucleic Acid Isolation kit. A different subset of specimen (4 normal liver specimens) were incubated at 95˚C for 30 min after protease digestion. DNA yield was determined by spectrophotometer and the number of amplifiable copies was determined by real-time PCR amplification of a 93 bp fragment of GAPDH.
Summary of Findings:
The yield of total DNA isolated from FFPE specimens did not appear to be adversely affected by the concentration of formalin (20% versus 10% formalin), fixation duration (2-9 d), or the temperature of fixation (25°C versus above 25°C). FFPE specimens fixed in 10% formalin had on average only 18% as much amplifiable GAPDH as case-matched frozen specimens, while specimens fixed in 20% formalin had only 0.3% as much amplifiable GAPDH as frozen specimens. The amount of amplifiable DNA decreased significantly when specimens were fixed for more than 3 days (in 10% formalin) compared to specimens fixed for 3 days or less (P<0.01). Formalin fixation at temperatures above 25˚C also reduced the amount of amplifiable DNA. Incubating a specimen at 95˚C for 30 min following protease digestion increased amplifiable DNA by a mean of 2.3-fold, but total DNA yield was unchanged.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 2 days
3 days
4 days
5 days
6 days
9 days
Biospecimen Preservation Concentration of fixative 10% formalin
20% formalin
Biospecimen Preservation Temperature of fixation/preservation ≤25˚C
>25˚C
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Analyte Extraction and Purification Incubation duration/condition 30 min incubation at 95˚C
Not heat-treated
Real-time qPCR Specific Targeted nucleic acid GAPDH
-
Study Purpose
This study included step-by-step analysis of the potential impact immunohistochemistry (IHC) staining on the yield of amplifiable and total DNA from FFPE specimens. Specimens used in this study included 4 FFPE normal liver specimens, two of which were fixed for 2 days, one for 3 days and the final for an unspecified duration. Additional details on formalin-fixation and paraffin embedding were not provided. FFPE sections (10 µm thick) from each of the FFPE normal liver specimens were subjected to varying numbers of steps in the immunohistochemical staining procedure. The steps in the immunostaining procedure included: a) sectioning, b) deparaffinization (method unspecified), c) antigen retrieval in 0.01 M citrate buffer, pH 6.0 at 120˚C for 15 min, d) peroxidase inactivation with 1% hydrogen peroxide in methanol, e) blocking in 5% skim milk for 30 min at 37˚C, f) incubation with anti-human hepatocyte antibody for 1 h followed by Histofine Simple Stain MAX PO for 30 min, g) colorization with 3,3'-diaminobenzidine (DAB). FFPE sections collected after each step were washed in distilled water and stored at -80˚C. Sections then underwent protease digestion overnight followed by DNA extraction using the RecoverAll Total Nucleic Acid Isolation kit for frozen FFPE sections. DNA yield was determined by spectrophotometer and the number of amplifiable copies was assessed by real-time PCR amplification of a 93 bp fragment of GAPDH.
Summary of Findings:
DNA yield and amount of amplifiable DNA decreased drastically after antigen retrieval to only approximately 20% of that obtained from an unstained section, but preceding and subsequent steps in the immunostaining procedure had little to no effect on the DNA yield or amount of amplifiable DNA. FFPE specimens yielded fragmented DNA, visible by electrophoresis, in comparison to DNA isolated from frozen specimens. The average fragment size also decreased in FFPE specimens subjected to antigen retrieval as part of the immunohistochemistry procedure.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
Formalin (buffered)
Analyte Extraction and Purification Antigen retrieval 0.01 M citrate buffer, pH 6.0 at 120˚C for 15 min
Not performed
Analyte Extraction and Purification Deparaffinization Deparaffinized
Not deparaffinized
Real-time qPCR Specific Targeted nucleic acid GAPDH
-
Study Purpose
This study compared next generation sequencing data obtained using case-matched FFPE and frozen normal liver specimens. Discrepancies between differentially fixed specimens were investigated further by Sanger sequencing or SYBR green allele-specific PCR. Details of specimen processing were not provided. After overnight protease digestion, DNA was extracted from frozen specimens using phenol-chloroform and from FFPE specimens using the RecoverAll Total Nucleic Acid Isolation kit. DNA yield was determined by spectrophotometer. Specimens were sequenced using the Ion AmpliSeq Comprehensive Cancer Panel on the Ion Proton sequencer and the Torrent Suite bioinformatics pipeline.
Summary of Findings:
Next generation sequencing of FFPE specimens identified 19 mutations not found in case-matched frozen specimens with a high confidence (P<1.0 x 10-7). However, further investigation by Sanger sequencing and allele-specific real-time PCR was not able to confirm three and six of the mutations, respectively. The authors concluded that the unconfirmed mutations identified by NGS were more likely an artifact of NGS than of the formalin fixation process.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA SNP assay DNA Real-time qPCR DNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Next generation sequencing Specific Technology platform Sanger sequencing
Real-time PCR
Biospecimen Preservation Type of fixation/preservation Frozen
Formalin (buffered)