Evaluation of DNA extraction from granulocytes discarded in the separation medium after isolation of peripheral blood mononuclear cells and plasma from whole bloodReport as inadecuate




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BMC Research Notes

, 6:440

First Online: 01 November 2013Received: 28 March 2013Accepted: 29 October 2013DOI: 10.1186-1756-0500-6-440

Cite this article as: Murray, J.R. & Rajeevan, M.S. BMC Res Notes 2013 6: 440. doi:10.1186-1756-0500-6-440

Abstract

BackgroundWhole blood is generally processed for plasma and peripheral blood mononuclear cells PBMCs from granulocytes-erythrocytes using gradient centrifugation of blood with Histopaue-Ficoll. After separation of plasma and PBMCs, the residual erythrocytes-granulocytes, a rich source of DNA, is often discarded along with the separation medium. In order to isolate DNA from the granulocytes, current methods require the removal of the separation medium and subsequent purification of granulocytes. This report provides a method for extracting DNA using the PAXgene Blood DNA kit from granulocytes without purifying them from the separation medium.

FindingsBased on 719 erythrocyte-granulocyte samples stored frozen for approximately 10 years in Ficoll-Hypaque separation medium, the mean yield of DNA was 395 μg median = 281 μg; range = 1.36 to 2077.2 μg, with mean A260-A280 ratio of 1.84 median = 1.84; range = 1.17 to 2.23. The quality of isolated DNA was sufficient for use as a template for restriction enzyme digestion, real-time PCR, pyrosequencing, and gel based variable number tandem repeats VNTR genotyping.

ConclusionsBy demonstrating the extraction of substantial amounts of high quality granulocytes DNA without purifying them from the separation medium, this method offers laboratories and biobanks a flexible and cost-effective approach to obtain plasma, PBMCs, and large amounts of DNA from a single blood collection for a variety of molecular genetics-epidemiologic studies.

KeywordsDNA extraction Granulocytes in separation medium Whole blood Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-6-440 contains supplementary material, which is available to authorized users.

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Author: Janná R Murray - Mangalathu S Rajeevan

Source: https://link.springer.com/







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