Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.
Author(s): Heikal N, Nussenzveig RH, Agarwal AM
Publication: Appl Immunohistochem Mol Morphol, 2014, Vol. 22, Page 623-6
PubMed ID: 24897067 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of FFPE block storage duration, deparaffinization method and duration of proteinase K digestion on DNA yield, and real-time PCR success and melting curve analysis from 12 formalin-fixed paraffin-embedded (FFPE) bone marrow trephine biopsies.
Conclusion of Paper
Comparable DNA yield, purity, mean fragment size, and PCR success rates were obtained from specimens deparaffinized with xylene and mineral oil, while those deparaffinized with HemoD were of a lower purity, had a smaller mean fragment size and a lower PCR success rate. The authors state that real-time PCR melting peaks were less homogeneous when specimens were deparaffinized with xylene than with mineral oil and that PCR success rates for HemoD-deparaffinized specimens declined with increasing storage duration. There was no effect of decreasing the proteinase K digestion duration from 72 h to 1 h for mineral oil-deparaffinized specimens with respect to DNA yield, purity, mean DNA fragment size, or PCR success rate.
Studies
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Study Purpose
The purpose of this study was to determine the effects of FFPE block storage duration and deparaffinization method on DNA yield, real-time PCR success and melting curve analysis from 12 FFPE bone marrow trephine biopsies. Specimens were stored as FFPE blocks for 5-19 years before DNA extraction. Five 5µm sections were deparaffinized by: i) two changes of xylene at 25°C for 15 min, ii) two changes of HemoD at an unspecified temperature for 5 min, or iii) mineral oil at 90°C for 20 min. After deparaffinization with xylene or HemoD specimens were rehydrated using a graded ethanol series before being treated with proteinase K for 72 h, but mineral oil deparaffinized specimens were directly digested with proteinase K for 72 h. DNA was extracted from all specimen using the QIAamp FFPE kit.
Summary of Findings:
Although it was possible to obtain 2-4 ng/µL DNA from five 5µm FFPE sections using each of the 3 deparaffinization methods, use of HemoD often resulted in lower yields and resulted in lower purity as determined by the ratio of the absorbance at 260 to 280 nm. Similarly, while the mean DNA fragment sizes for mineral oil- or xylene-deparaffinized specimens were 188 and 174 bp, respectively, the mean DNA fragment size for HemoD-deparaffinized specimens was only 71 bp. The authors report a 100% PCR success rate when specimens were deparaffinized using mineral oil or xylene, but they report a lower PCR success rate for HemoD-deparaffinized specimens and state that most failures occurred in specimens stored for >10 years. Finally, the authors state that real-time PCR melting curves were less homogeneous when specimens were deparaffinized with xylene than with mineral oil.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 5-19 years
Analyte Extraction and Purification Deparaffinization Two changes of 25°C xylene for 15 min
90°C mineral oil for 20 min
Two changes of HemoD each for 5 min
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Study Purpose
The purpose of this study was to determine the effects of proteinase K digestion duration and FFPE block storage duration on DNA yield and real-time PCR success from 12 FFPE bone marrow trephine biopsies. Specimens were stored for 5-19 years before DNA extraction. Five 5µm sections were deparaffinized by incubation in 90°C mineral oil for 20 min. After deparaffinization, specimens were treated with proteinase K for 72 h or 1 h. DNA was extracted from all specimen using the QIAamp FFPE kit.
Summary of Findings:
For mineral oil-deparaffinized specimens, decreasing the duration of proteinase K digestion from 72 h to 1 h had no effect on DNA yield, purity or mean DNA fragment size. Further, the PCR success rate was 100%, regardless of storage duration and digestion duration.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 5-19 years
Analyte Extraction and Purification Protein digestion 1 h
72 h