Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.
Author(s): Steinau M, Patel SS, Unger ER
Publication: J Mol Diagn, 2011, Vol. 13, Page 377-81
PubMed ID: 21704270 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of deparaffinization and lysis temperature on DNA yield, and quantification and genotyping of HPV in cervical, vaginal, anal, penile, tonsil and laryngeal SCC specimens. After deparaffinization (heat treatment or xylene) and lysis with proteinase K, DNA was extracted using DNeasy from two 5 um sections of FFPE blocks stored 2-4 years.
Summary of Findings:
The heat-treatment deparaffinization protocol yielded 157 pg/uL DNA which contained 1639 copies HPV 16 per mL, but the xylene treatment protocol only yielded 23 pg/uL DNA (p<0.001) and 405 copies HPV 16 per mL (p<0.009). HPV genotyping by linear array was not possible from 29/150 xylene-deparaffinized specimens lysed at 56 degrees C and 8/150 specimens that were heat treated and lysed at 65 degrees C (p=0.0003). HPV genotype concordance, by linear array, between extraction methods was 99.3% with 86.5% of the discordance due to additional types being detected in heat-treated specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Lysis at 56 degrees C
Lysis at 65 degrees C
Analyte Extraction and Purification Deparaffinization Xylene
20 min at 120 degrees C in ATL buffer
Real-time qPCR Specific Targeted nucleic acid Viral L1 region of HPV16
Beta-globin