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

Abstract

Aim-To evaluate the eYciency of phenol/ chloroform, microwave, and Qiagen spin column based DNA extractions from paraYn 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 globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results-Microwave extraction showed the highest positive rate for globin PCR, whereas the spin column method was the most eYcient for HPV DNA extraction. When the 509 bp globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 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 globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions-HPV DNA extraction was most eYcient 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 eYcient. 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. (J Clin Pathol 2001;54:401-403) Keywords: cervical cancer; DNA extraction; polymerase chain reaction ParaYn wax embedded tissues are a valuable source of DNA for analysis. The traditional method of purifying DNA from archival tissues using xylene dewaxing followed by phenol/ chloroform purification is labourious and requires multiple steps that are prone to cross contamination. Recently, alternative approaches have been used. [1][2][3] In most studies, the success of DNA extraction is assessed by amplifying a fragment of a housekeeping gene, and the number of positive samples are regarded as a denominator when calculating the prevalence of target pathogens. Whether such an assessment can guarantee the retention of an equivalent length of DNA of the target pathogen has not been fully elucidated. In our study, three methods were applied on a series of paraYn wax embedded tissues to compare their eYciency for purifying human and viral DNA, and to assess the reliability of using a housekeeping gene as an indicator for successful viral DNA extraction. Methods STUDY SAMPLESTwenty blocks of formalin fixed, paraYn wax embedded cervical squamous cell carcinoma stored for four to six years were analysed. For each case, four 5 µm thick slices were cut in triplicate. The area to be sectioned was examined carefully to ensure the inclusion of tumour tissue and that an equal amount of tissue was included in each set. Three diVerent DNA purification methods were performed for each case. Thorough cleansing was performed between cases and a new set of cutting instruments ...

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936

“…Numerous protocols for the extraction of DNA from paraffin-embedded tissue for PCR analysis have been published. 181,182,[185][186][187] Many of these aim to reduce DNA degradation and the coextraction of PCR inhibitors, but many of these methods require prolonged extraction procedures and can be unsuitable for use in the routine diagnostic laboratory. 176,177,188,189 DNA sample concentration and integrity were estimated by spectrophotometry and by comparison of sample DNA with known standards on agarose-gel electrophoresis.…”

Biases in human papillomavirus genotype prevalence assessment associatedwith commonly used consensus primers

“…150 bp fragment of the L1 region. 5,6 The HPV type was identified by direct sequencing of PCR amplicons. In the second method, the MY09/ 11 primers that target an approx.…”

“…Total DNA was extracted by the QIAamp DNA mini kit (QIAGEN, Hilden, Germany) and with the quality of extracted preparations confirmed by beta-globin PCR. 5 The clinical materials were collected with a written informed consent. Our study was approved by the local institutional ethics committee, and the human experimentation guidelines of the local institute were followed in the conduct of our study.…”

Distribution of human papillomavirus types in cervical cancers in Hong Kong: Current situation and changes over the last decades

“…The DNA quality of paraffin-embedded tissues was assessed by a PCR targeting a 355-bp fragment of the house-keeping gene beta-globin. 19 The presence of HPV DNA was detected by the INNO-LiPa HPV Genotyping Extra kit (Innogenetics, Belgium), which can identify 27 HPV types (HPV6, 11,16,18,26,31,33,35,39,40,43,44,45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73 and 82). The INNO-LiPa kit was selected for paraffin-embedded tissues because it targets a shorter fragment (65 bp) that can usually be recovered from formalin-fixed tissues.…”

“…DNA extraction from paraffin-embedded tissues was carried out according to our previously described method. 19 Briefly, the tissues were first dewaxed with 2 washes of xylene, and then subjected to DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Hilden). The DNA quality of paraffin-embedded tissues was assessed by a PCR targeting a 355-bp fragment of the house-keeping gene beta-globin.…”