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KRAS mutation analysis by PCR : a comparison of two methods
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Bolton, Louise, Reiman, Anne, Lucas, Katie, Timms, Judith and Cree, Ian A. (2015) KRAS mutation analysis by PCR : a comparison of two methods. PLoS One, Volume 10 (Number 1). Article number e0115672. doi:10.1371/journal.pone.0115672 ISSN 1932-6203.
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Official URL: http://dx.doi.org/10.1371/journal.pone.0115672
Abstract
Background:
KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.
Methods:
DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer’s instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.
Results:
Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.
Conclusion:
There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.
| Item Type: | Journal Article | ||||||||||
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| Subjects: | Q Science > QH Natural history | ||||||||||
| Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Translational & Experimental Medicine > Reproductive Health ( - until July 2016) Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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| Library of Congress Subject Headings (LCSH): | Colon (Anatomy) -- Cancer, Polymerase chain reaction | ||||||||||
| Journal or Publication Title: | PLoS One | ||||||||||
| Publisher: | Public library of science | ||||||||||
| ISSN: | 1932-6203 | ||||||||||
| Official Date: | 8 January 2015 | ||||||||||
| Dates: |
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| Volume: | Volume 10 | ||||||||||
| Number: | Number 1 | ||||||||||
| Number of Pages: | 13 | ||||||||||
| Article Number: | Article number e0115672 | ||||||||||
| DOI: | 10.1371/journal.pone.0115672 | ||||||||||
| Status: | Peer Reviewed | ||||||||||
| Publication Status: | Published | ||||||||||
| Access rights to Published version: | Open Access (Creative Commons) | ||||||||||
| Date of first compliant deposit: | 28 December 2015 | ||||||||||
| Date of first compliant Open Access: | 28 December 2015 | ||||||||||
| Funder: | Life Technologies, Merck Serono |
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