Hanspal Singh1, Bhaskar Narayan2, Aadithya B Urs3, Sunil Kumar Polipalli4, Somesh Kumar5. 1. Senior Research Associate (Oral Pathology & Microbiology), Maulana Azad Institute of Dental Sciences, New Delhi, India. 2. Post Graduate Student (Oral Pathology & Microbiology), Maulana Azad Institute of Dental Sciences, New Delhi, India. 3. Professor & Head (Oral Pathology & Microbiology), Maulana Azad Institute of Dental Sciences, New Delhi, India. 4. Cytogeneticist (Pediatrics Research & Genetics Laboratory), Maulana Azad Medical College, New Delhi, India. 5. Ph.D Scholar (Pediatrics Research & Genetics Laboratory), Maulana Azad Medical College, New Delhi, India.
Abstract
BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue is the gold standard procedure for tissue preservation. However, the extraction of DNA is a cumbersome procedure as the extracted DNA is fragmented because of the cross-linking effect of formalin. Hence, the aim of the present study is to extract DNA from FFPE tissues using different techniques with a specific objective of comparing the extracted DNA, both quantitatively and qualitatively. METHOD: Ten samples of FFPE tissues were retrieved from the archives of the Department of Oral Pathology. Total genomic DNA was extracted by different methods which included QIAamp DNA FFPE Tissue Kit, Norgen DNA FFPE Tissue Kit, phenol-chloroform method, mineral oil-based extraction, M/10 NaOH solution method, and microwave method. A 280-base pair sequence was selected for evaluation of downstream amplification. RESULTS: The statistical analysis was performed using unpaired student's t-test to compare the DNA yields and quality obtained by microwave methods with other methods using SPSS software. Total genomic DNA retrieved by the microwave method was superior to other methods both qualitatively and quantitatively. CONCLUSION: DNA extraction from FFPE tissues is an onerous task as irreversible bonds form between the nucleic acid during fixation which are difficult to break during DNA retrieval. Hence, the microwave method provides good total genomic DNA which gives better downstream results when compared with other methods.
BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue is the gold standard procedure for tissue preservation. However, the extraction of DNA is a cumbersome procedure as the extracted DNA is fragmented because of the cross-linking effect of formalin. Hence, the aim of the present study is to extract DNA from FFPE tissues using different techniques with a specific objective of comparing the extracted DNA, both quantitatively and qualitatively. METHOD: Ten samples of FFPE tissues were retrieved from the archives of the Department of Oral Pathology. Total genomic DNA was extracted by different methods which included QIAamp DNA FFPE Tissue Kit, Norgen DNA FFPE Tissue Kit, phenol-chloroform method, mineral oil-based extraction, M/10 NaOH solution method, and microwave method. A 280-base pair sequence was selected for evaluation of downstream amplification. RESULTS: The statistical analysis was performed using unpaired student's t-test to compare the DNA yields and quality obtained by microwave methods with other methods using SPSS software. Total genomic DNA retrieved by the microwave method was superior to other methods both qualitatively and quantitatively. CONCLUSION: DNA extraction from FFPE tissues is an onerous task as irreversible bonds form between the nucleic acid during fixation which are difficult to break during DNA retrieval. Hence, the microwave method provides good total genomic DNA which gives better downstream results when compared with other methods.
Authors: R W M Hoetelmans; H J van Slooten; R Keijzer; C J H van de Velde; J H van Dierendonck Journal: Biotech Histochem Date: 2002-05 Impact factor: 1.718
Authors: Bernard Metz; Gideon F A Kersten; Peter Hoogerhout; Humphrey F Brugghe; Hans A M Timmermans; Ad de Jong; Hugo Meiring; Jan ten Hove; Wim E Hennink; Daan J A Crommelin; Wim Jiskoot Journal: J Biol Chem Date: 2003-11-24 Impact factor: 5.157