Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA

摘要

Aim—To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. Methods—DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the ß globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results—Microwave extraction showed the highest positive rate for ß globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp ß globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 ß globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the ß globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions—HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered. >Key Words: cervical cancer • DNA extraction • polymerase chain reaction