Deparaffinization and lysis by hydrothermal pressure (pressure cooking) coupled with chaotropic salt column purification: a rapid and efficient method of DNA extraction from formalin-fixed paraffin-embedded tissue.
We report a hydrothermal pressure method (pressure cooking) for simultaneous deparaffinization and lysis of formalin-fixed paraffin-embedded tissue followed by conventional chaotropic salt column purification to obtain high-quality DNA. Using this method, the release of DNA occurred within the first minute of treatment, reaching the maximum at 5 minutes. An optimal treatment window was between 5 and 30 minutes. The extracted DNA was of high quality as determined by the 260/280 absorbance ratios, and the quantity of DNA extracted was linear with the input tissue amount. In paired sample experiments (N=19), the quantity of DNA extracted by hydrothermal pressure treatment was comparable to that obtained through the conventional xylene deparaffinization and protease K digestion method. The integrity of the recovered DNA was also comparable, evidenced by polymerase chain reaction amplifications of variable-sized amplicons in tissue samples archived from 0.2 to 22 years (N=14). The high quality of DNA was further confirmed by polymerase chain reaction amplification and Sanger sequencing analysis of representative exons of the EGFR gene in human non-small cell lung cancer tissue samples. In summary, this novel method offers DNA release from formalin-fixed paraffin-embedded tissue with unprecedented simplicity, speed, biohazard safety, and cost-efficiency. Combined with chaotropic salt column purification, high-quality DNA can be prepared for downstream applications in <30 minutes.
Department of Pathology, Health Science Center, Peking University, Beijing, China.
Haohao
H
Zhong
Yan
Y
Liu
Monica
M
Talmor
Bingquan
B
Wu
Pei
P
Hui
D003296:Cooking / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D005557:Formaldehyde; D006358:Hot Temperature; D006801:Humans; D006874:Hydrostatic Pressure; D010232:Paraffin; D057089:Pathology, Molecular / Q000379:methods*; D012492:Salts; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D016610:Tissue Embedding; D016707:Tissue Fixation
D012492:Salts; D005557:Formaldehyde; D010232:Paraffin; D004247:DNA