Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec;14(6):520-529.
doi: 10.1089/bio.2016.0011. Epub 2016 Aug 22.

Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood

Affiliations

Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood

Conny Mathay et al. Biopreserv Biobank. 2016 Dec.

Abstract

Background: This article is the fifth in a series of publications providing formal method validation for biospecimen processing. We report the optimization and validation of methodology to obtain nucleic acids of sufficient quantity and quality from blood.

Methods: DNA was extracted using the Chemagic DNA Blood Kit on an MSM I. Extraction was optimized in terms of blood volume, elution buffer volume, and lysis conditions. The optimal protocol was validated for reproducibility, robustness (delay to buffy coat extraction, blood vs. buffy coat, and use of a magnetic rack), and performance (yield, purity, and concentration). RNA was extracted using a PAXgene Blood miRNA kit with a QiaCube. The protocol was validated for reproducibility, robustness (elution buffer, delay, and temperature before extraction), and performance (yield, purity, integrity, and miRNA content). Two platforms (QiaCube, Biorobot Universal) were further compared.

Results: For DNA extraction, a 4 mL blood sample, manual lysis, and 300 μL elution buffer were found to be reproducible (CV <10% for DNA yield and A260 nm/A280 nm ratio) and robust (buffy coat vs. whole blood; immediate processing of buffy coat after lysis vs. storage for 1 week at 2-8°C; and magnetic rack use). There was no difference between automated and manual lysis. RNA extracted with the PAXgene Blood miRNA kit on a QiaCube gave high yields and optimal reproducibility (low CV for RNA yield and integrity) with BR5 elution buffer (vs. water and TE). PAXgene tubes could be stored for up to 2 weeks at 2-8°C. The Biorobot Universal System gave similar mean RNA yields with Qiacube and slightly lower but acceptable purity.

Conclusions: We validated automated isolation of DNA with a Chemagic DNA Blood Kit on a magnetic bead-based MSM I, and of RNA with a PAXgene Blood miRNA kit on a silica membrane-based QiaCube or Biorobot (for low and high throughput, respectively).

Keywords: DNA; RNA; method validation; processing method; whole blood.

PubMed Disclaimer

Similar articles

Cited by

LinkOut - more resources