Preparation of uniform, intact DNA samples from resected tumor tissues for pulsed-field gel electrophoretic analyses.
Preparation of high molecular weight DNA from resected tumor tissues suitable for pulsed-field gel electrophoresis (PFGE) can be complicated by the presence of nonviable cells and lymphocytes. We have developed a simple procedure to reduce the level of degraded DNA in PFGE DNA samples prepared from resected tumor tissues. The procedure employs a single, three component Percoll step gradient centrifugation and can be performed on several tumor samples simultaneously. Analyses of DNAs from 15 tumor specimens (7 solid tumors and 8 aspirated fluids) demonstrate that the technique enriches the integrity of PFGE DNA samples. Morphologic evaluation of 9 specimens suggested that both cellular debris and contaminating normal lymphocytes are removed from starting cell populations during the enrichment procedure. Fractionation of cells also reduced cell clumping, allowing for the formation of more uniform PFGE DNA samples.
VanDevanter
D R
DR
Tumor Institute, Swedish Hospital Medical Center, Seattle, WA 98104.
Yirdaw
G
G
Do
C
C
Tysseling
K A
KA
Drescher
C A
CA
Forseth
B J
BJ
Von Hoff
D D
DD
McNutt
M A
MA
D002499:Centrifugation, Density Gradient; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D005069:Evaluation Studies as Topic; D006801:Humans; D008970:Molecular Weight; D009369:Neoplasms / Q000737:chemistry / Q000473:pathology; D011205:Povidone; D012822:Silicon Dioxide
D004273:DNA, Neoplasm; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone