Zainabadi, Kayvan and Dhayabaran, Vaigundan and Moideen, Kutty and Krishnaswamy, Patnam (2019) An efficient and cost-effective method for purification of small sized DNAs and RNAs from human urine. In: PLOS ONE, 14 (2).
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Abstract
Urine holds great promise as a non-invasive sampling method for molecular diagnostics. The cell-free nucleic acids of urine however are small, labile, and difficult to purify. Here an efficient method for the purification of these nucleic acids is presented. An empirically derived protocol was devised by first identifying conditions that allowed recovery of a 100 base pair (bp) DNA, followed by optimization using a quantitative polymerase chain reaction (qPCR) assay. The resulting method efficiently purifies both small sized DNAs and RNAs from urine, which when combined with quantitative reverse transcription PCR (qRTPCR), demonstrably improves detection sensitivity. Fractionation experiments reveal that nucleic acids in urine exist both in the cell-free and cellular fraction, roughly in equal proportion. Consistent with previous studies, amplicons > 180bp show a marked loss in PCR sensitivity for cell-free nucleic acids. Finally, the lysis buffer developed here also doubles as an effective preservative, protecting against nucleic acid degradation for at least two weeks under simulated field conditions. With this method, volumes of up to 25ml of whole urine can be purified in a high-throughput and cost-effective manner. Coupled with its ability to purify both DNA and RNA, the described method may have broad applicability for improving the diagnostic utility of urine, particularly for the detection of low abundant targets.
| Item Type: | Journal Article |
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| Publication: | PLOS ONE |
| Publisher: | PUBLIC LIBRARY SCIENCE |
| Additional Information: | Copyright of this article belongs to PUBLIC LIBRARY SCIENCE |
| Department/Centre: | Division of Biological Sciences > Biochemistry |
| Date Deposited: | 19 Feb 2019 05:32 |
| Last Modified: | 19 Feb 2019 05:32 |
| URI: | http://eprints.iisc.ac.in/id/eprint/61750 |
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