Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues.
Isolation of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue remains a laborious task for clinical laboratories and researchers who need to screen several samples for genetic variants. The objective of this study was to evaluate DNA isolation methods from FFPE tissues and to choose an efficient method with less hands-on time to obtain DNA of optimum concentration and purity for use in routine molecular diagnostic assays. Three methods were compared in this study: Gentra Puregene Tissue Kit, EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. Samples consisted of FFPE tissues of head/neck and lung tumor resections. Quality control for the extraction process end product included determination of the concentration and purity of isolated DNA and the ability to amplify a housekeeping gene, GAPDH, using real-time PCR assay. The hands-on-time required was less for the EZ1 protocol compared to the other methods. The average DNA concentration obtained was 112, 61 and 40 ng/μl, respectively, for the Gentra Puregene Tissue Kit, Qiagen EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. The purity and quality of samples obtained using the different DNA isolation methods were comparable. Comparative evaluation of three DNA isolation methods indicated that the Qiagen EZ1 method surpassed the other methods with reduced hands-on-time to produce optimum concentration of quality DNA for use in routine molecular analyses.
Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, United States.
Soya S
SS
Sam
Kimberly A
KA
Lebel
Cheryl L
CL
Bissaillon
Laura J
LJ
Tafe
Gregory J
GJ
Tsongalis
Joel A
JA
Lefferts
D001331:Automation; D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde; D016678:Genome; D015894:Genome, Human; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006258:Head and Neck Neoplasms / Q000737:chemistry / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000175:diagnosis / Q000235:genetics; D016612:Paraffin Embedding; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013997:Time Factors; D016707:Tissue Fixation
D011933:Reagent Kits, Diagnostic; D005557:Formaldehyde; D004247:DNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)