Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA.
J Transl Med. 2011 Jun 10;9:91. doi: 10.1186/1479-5876-9-91.
Blood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping.
We isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis.
We tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%).
When collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80°C frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing.
在早期研究访问中采集血液标本常用于分析次要结局生物标志物的观察性研究或临床试验。一种常见的方案是储存用于未来 DNA 分离的白细胞层标本，这些标本可能在冷冻储存中保存多年。目前尚不确定这些 DNA 是否仍然适合现代全基因组关联 (GWA) 基因分型。
我们从 120 例行动控制心血管风险糖尿病 (ACCORD) 临床试验的白细胞层中分离 DNA，这些标本的储存时间从接近 9 年到其他可能影响 DNA 产量的因素不等。我们进行了 TaqMan SNP 和 GWA 基因分型，以测试 DNA 是否保持完整性以进行高质量的遗传分析。
我们测试了两种 QIAGEN 自动化 DNA 分离方案，尽管产量相似，但更喜欢受损血液方案。我们从所有 120 个标本中分离出 DNA（产量范围为每 8.5 毫升 ACD 管全血 1.1-312 微克），只有 3/120 个样本的 DNA 产量<10 微克。采血时参与者的年龄与产量呈负相关（平均变化量为-2.1 微克/年）。根据凝胶电泳 QC、6 个 SNP 的 TaqMan 基因分型（702 个基因型中基因分型无呼叫率为 1.1%）和出色的 GWA 基因分型数据（每个样本基因型缺失率最高为 0.64%），DNA 质量非常好。
当作为长期临床试验或生物库标本采集 DNA 时，白细胞层可在-80°C 的冷冻状态下储存长达 9 年，仍然可以产生适合 GWA 分析和其他遗传测试的高产量 DNA。
