Deparaffinization of formalin-fixed paraffin-embedded tissue blocks using hot water instead of xylene.
Author(s): Kalantari N, Bayani M, Ghaffari T
Publication: Anal Biochem, 2016, Vol. 507, Page 71-3
PubMed ID: 27287960 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of deparaffinization in hot water rather than xylene and DNA extraction kit type on the yield and purity of DNA isolated from archived formalin-fixed paraffin-embedded (FFPE) breast and lymph specimens and determined if hot water deparaffinization was compatible with PCR.
Conclusion of Paper
Deparaffinization in water had no effect on the yield or purity of the extracted DNA nor did it affect PCR amplification of a 185 bp fragment of toll-like receptor 4 (TLR4). Extraction with the Bio Basic One-Tube DNA Extraction kit resulted in higher DNA yields and purity than extraction with the Yekta Tajhiz DNA extraction mini kit.
Studies
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Study Purpose
This study investigated the effects of deparaffinization in hot water rather than xylene and DNA extraction kit type on the yield and purity of DNA isolated from archived FFPE breast and lymph specimens and determined if hot water deparaffinization was compatible with PCR. Seven (7) µm sections were cut from 25 archival breast and lymph specimens of unspecified age and diagnosis. Sections were deparaffinized in water through the addition of 1 mL of 90˚C water and after the paraffin was completely melted, the specimen was washed two more times with 90˚C water. Ten specimens were deparaffinized in xylene by two 15 min room temperature incubations in 1.5 mL xylene, followed by washing in ethanol, and rehydration in distilled water. DNA was extracted using the Bio Basic One-Tube DNA Extraction kit or the Yekta Tajhiz DNA extraction mini kit. DNA yield and purity were assessed spectrophotometrically.
Summary of Findings:
The quantity and purity of DNA isolated from specimens deparaffinized by the hot water method was comparable to that in specimens deparaffinized with xylene. Further, amplification of a 185 bp fragment of TLR4 was possible, regardless of deparaffinization method. Extraction with the Bio Basic One-Tube DNA Extraction kit resulted in higher DNA yields and purity than extraction with the Yekta Tajhiz DNA extraction mini kit, but the significance of the difference was not presented.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA PCR DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Deparaffinization 90˚C water
Xylene
PCR Specific Targeted nucleic acid TLR4
Analyte Extraction and Purification Analyte isolation method Yekta Tajhiz DNA extraction mini kit
Bio Basic One-Tube DNA Extraction kit