Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA.
OBJECTIVE
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 beta globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA.


RESULTS
Microwave extraction showed the highest positive rate for beta globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp beta globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 beta 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 beta 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.
Chan
P K
PK
Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. paulkschan@cuhk.edu.hk
Chan
D P
DP
To
K F
KF
Yu
M Y
MY
Cheung
J L
JL
Cheng
A F
AF
D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000821:virology*; D002725:Chloroform; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D005914:Globins / Q000235:genetics; D006801:Humans; D008872:Microwaves; D027383:Papillomaviridae / Q000145:classification / Q000302:isolation & purification*; D016612:Paraffin Embedding; D019800:Phenol; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000821:virology*
D004273:DNA, Neoplasm; D004279:DNA, Viral; D019800:Phenol; D002725:Chloroform; D005914:Globins