Removal of high-molecular-weight DNA by carboxylated magnetic beads enhances the detection of mutated K-ras DNA in urine.
Author(s): Su YH, Song J, Wang Z, Wang XH, Wang M, Brenner DE, Block TM
Publication: Ann N Y Acad Sci, 2008, Vol. 1137, Page 82-91
PubMed ID: 18837929 PubMed Review Paper? No
Purpose of Paper
This paper compared the size profile of DNA that was isolated from whole urine as well as fromurine cellular debris and urine supernatant; urine was collected from six patients at three different times of the day. The authors also investigate if KRAS mutation detection in urine can be increased by selective removal of high molecular weight DNA using a carboxylated magnetic bead-based method and urine specimens from patients with colorectal cancer, adenomatous polyps, hyperplastic polyps, or without a known neoplasia.
Conclusion of Paper
High and low molecular weight DNA were present in all specimens regardless of whether they were whole urine speciens or the cellular debris pellet or supernatant, but the amount of DNA and its relative abundance differed among patients and collection times, although there was no observable pattern . The carboxylated magnetic bead-based method was able to remove an average of 97.2% of the high molecular weight DNA. Removal of the high molecular weight DNA increased the detection rate of the KRAS mutation from 43.8% to 87.5% in specimens from colorectal cancer patients and from 16.7 to 75% in specimens from patients with adenomatous polyps. Importantly, concordance in KRAS mutation detection between matched urine and tissue sections increased from 38% to 86% after removal of high molecular weight DNA (P=0.015). The authors conclude that removal of high molecular weight DNA increases the proportion of mutated DNA,which allows for better detection.
Studies
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Study Purpose
This study compared the size profile of DNA that was isolated from whole urine as well as from urine cellular debris and urine supernatant; urine was collected from six patients at three different times of the day. The authors also investigate if KRAS mutation detection in urine can be increased by selective removal of high molecular weight DNA using a carboxylated magnetic bead-based method and specimens from patients with colorectal cancer, adenomatous polyps, hyperplastic polyps or without a known neoplasia. For all studies, urine was collected and immediately mixed with EDTA (pH 8.0) to achieve a concentration of 10 mM. Urine was then stored at -70°C. Urine was thawed at room temperature and placed on ice for < 1 h before DNA isolation with the Wizard DNA Isolation Kit. DNA was extracted from paraffin-embedded tissue sections (no additional details were provided) using the MasterPure DNA Kit. For analysis of variability and collection timing effects, urine was collected from six patients immediately after waking, at 10 AM, and at 2 PM. DNA was extracted from whole urine and from the resulting supernatant and cellular debris pellet after centrifugation at 2000 rpm for 10 min. DNA was characterized by electrophoresis. To investigate the ability of an in-house carboxylated magnetic bead-based method to remove high molecular weight DNA, long and short DNA were quantified by real-time PCR using DNA from the urine of six volunteers before and after treatment with magnetic beads. To test the effect of removal of high molecular weight DNA on the detection of KRAS mutations, KRAS mutations were detected by restriction endonuclease-enriched PCR in matched specimens of urine and tissue sections from 36 patients (16 with colorectal cancer, 12 with adenomatous polyps, 2 with hyperplastic polyps and 7 without a neoplasia) before and after removal of high molecular weight DNA.
Summary of Findings:
Both high and low molecular weight DNA were present in all specimens regardless of whether DNA was from whole urine, the cellular debris pellet, or the supernatant, but the amount of DNA and the relative abundance differed among patients and collection times; although, there was no clear pattern among collection times. The carboxylated magnetic bead-based method was able to remove an average of 97.2% (88.1-96%) of high molecular weight DNA from six whole urine specimens. Removal of the high molecular weight DNA increased the detection of the KRAS mutation from 43.8% to 87.5% in whole urine specimens from colorectal cancer patients (P=0.0233) and from 16.7 to 75% in specimens from patients with adenomatous polyps (P=0.0123). Importantly, concordance in KRAS mutation detection between matched urine and tissue sections increased from 38% to 86% after removal of high molecular weight DNA (P=0.015). KRAS mutations were only detected in the urine and tissue of one of the two patients with hyperplastic polyps after removal of high molecular weight DNA, with no detection in either tissue or urine of the other patient. One of the seven urine specimens from patients without a neoplasias also tested positive for KRAS mutations after removal of high molecular weight DNA, but there was no matching tissue specimen. The authors conclude that removal of high molecular weight DNA increases the proportion of mutated DNA, which allows for superior detection of KRAS mutations.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Neoplastic - Benign
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Restriction fragment length polymorphism DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Restriction fragment length polymorphism Specific Template modification No removal of high molecular weight DNA
Removal of high molecular weight DNA
Preaquisition Diagnosis/ patient condition Colorectal carcinoma
Adenomatous polyps
Hyperplastic polyps
No known neoplasia
Biospecimen Aliquots and Components Biospecimen components Whole urine
Urine cell debris
Urine supernatant
Biospecimen Aliquots and Components Centrifugation Centrifuged
Not centrifuged
Biospecimen Acquisition Type of collection container/solution First void
10 AM
2 PM