Details:

Alternative Title:
BMC Res Notes
Publisher's site:
http://dx.doi.org/10.1186/1756-0500-6-440
Personal Author:
Murray, Janná R ; Rajeevan, Mangalathu S ;
Murray, Janná R ; Rajeevan, Mangalathu S ; Less -
Description:
Background Whole 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. Findings Based 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. Conclusions By 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.
Subject:
[+]
Cell Separation
Centrifugation
DNA
DNA Extraction
Genotyping Techniques
Granulocytes
Granulocytes In Separation Medium
Humans
Leukocytes, Mononuclear
Minisatellite Repeats
Reagent Kits, Diagnostic
Real-Time Polymerase Chain Reaction
Restriction Mapping
Sequence Analysis, DNA
Short Report
Whole Blood

Source:
BMC Res Notes. 2013; 6:440.
Document Type:
Journal Article ;
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:85e7d7278a58208888e2724f98b818ac536af1b7cd2edc4efd9c0a64c5b7f0d7
File Type:

Supporting Files

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