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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 PLoS+One
2018 ; 13
(2
): e0190280
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DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic
kidney disease: A pilot study
#MMPMID29462136
Lecamwasam A
; Sexton-Oates A
; Carmody J
; Ekinci EI
; Dwyer KM
; Saffery R
PLoS One
2018[]; 13
(2
): e0190280
PMID29462136
show ga
AIM: To characterise the genomic DNA (gDNA) yield from urine and quality of
derived methylation data generated from the widely used Illuminia Infinium
MethylationEPIC (HM850K) platform and compare this with buffy coat samples.
BACKGROUND: DNA methylation is the most widely studied epigenetic mark and
variations in DNA methylation profile have been implicated in diabetes which
affects approximately 415 million people worldwide. METHODS: QIAamp Viral RNA
Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh
urine samples as well as increasing volumes of fresh urine. Matched buffy coats
to the frozen urine were also obtained and DNA was extracted from the buffy coats
using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20?g/ml
were used for methylation analysis using the HM850K array. RESULTS: Irrespective
of extraction technique or the use of fresh versus frozen urine samples, limited
genomic DNA was obtained using a starting sample volume of 5ml (0-0.86?g/mL). In
order to optimize the yield, we increased starting volumes to 50ml fresh urine,
which yielded only 0-9.66?g/mL A different kit, QIAamp DNA Micro Kit, was
trialled in six fresh urine samples and ten frozen urine samples with inadequate
DNA yields from 0-17.7?g/mL and 0-1.6?g/mL respectively. Sufficient genomic DNA
was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA
methylation profiling. In comparison, all four buffy coat samples (100%) provided
sufficient genomic DNA. CONCLUSION: High quality data can be obtained provided a
sufficient yield of genomic DNA is isolated. Despite optimizing various
extraction methodologies, the modest amount of genomic DNA derived from urine,
may limit the generalisability of this approach for the identification of DNA
methylation biomarkers of chronic diabetic kidney disease.