Abstract
ObjectiveIn the present study, we investigated different simple and cost effective methods to evaluate and validate cell free DNA (cfDNA) isolation. The ability of the QIAamp DNA Blood Mini Kit method to extract cfDNA was assessed by several approaches, including purification of endogenous cfDNA and exogenous spike-in control material, prior to plasma extraction, and followed by quantitative-PCR.ResultsUsing QIAamp DNA Blood Mini kit, nearly 27% (380 bp) to 35% (173 bp) cfDNA was recovered with a higher recovery of smaller size cfDNA (173 bp) in comparison to larger ones (380 bp). These simple laboratory methods can be used to assess the efficiency of any cfDNA isolation method.
Highlights
Circulating cell-free DNA molecules are shed into bloodstream, plasma, serum, urine as well as other body fluids of humans [1]
Experiment 4: following spiked male genomic DNA To measure the absolute quantification of cell-free DNA (cfDNA) extracted by Qiagen method, pooled plasma of healthy woman was divided in 11 microtubes (300 μl in each)
Experiment 4: result CfDNA extraction for Qiagen method were performed in 10 replicates and each sample was quantified by SYBRGreen quantitative PCR (qPCR) in triplicate for DAZ locus (380 bp) and DYS221 locus (173 bp) separately
Summary
Circulating cell-free DNA (cfDNA) molecules are shed into bloodstream, plasma, serum, urine as well as other body fluids of humans [1]. PCR was carried out in a 15 μl total volume using HiFi SYBR Green Master Mix (Farabin, Iran), 300 nM of each primer (Additional file 1: Table S1) , and 2.5 μl of DNA template.
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