Knudsen Beatrice S, Allen April N, McLerran Dale F, Vessella Robert L, Karademos Jonathan, Davies Joan E, Maqsodi Botoul, McMaster Gary K, Kristal Alan R
Fred Hutchinson Cancer Research Center, Division of Public Health Sciences, Seatle, Washington, USA.
J Mol Diagn. 2008 Mar;10(2):169-76. doi: 10.2353/jmoldx.2008.070127. Epub 2008 Feb 14.
We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.
我们评估了分支DNA（bDNA）检测法QuantiGene试剂系统，以测量福尔马林固定、石蜡包埋（FFPE）组织中的RNA。QuantiGene试剂系统无需进行RNA分离，避免了酶促预扩增，且工作流程简单。通过bDNA检测法测定了五个选定基因；定量聚合酶链反应（qPCR）用作参考方法。使用混合效应统计模型将总体方差划分为可归因于异种移植、样本和检测的成分。对于FFPE组织，bDNA检测法的可靠性系数（93 - 100%）显著高于qPCR（82.4 - 95%）。qPCR（冷冻样本，金标准）与bDNA（FFPE样本）之间的相关性在0.60至0.94之间，与qPCR（冷冻样本）和qPCR（FFPE样本）之间的相关性相似。此外，bDNA检测法在组织匀浆中的灵敏度比在纯化RNA中高10倍。在RNA质量较差的9至13年的样本块中，bDNA检测法能够正确识别已知癌症基因的过表达。总之，QuantiGene试剂系统比qPCR更可靠、可重复且灵敏得多，为测量FFPE组织中的基因表达提供了一种替代方法。它似乎也非常适合用于患者治疗和管理的具有诊断或预后基因表达生物标志物面板的FFPE组织的临床分析。
