A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples.
A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples.
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DOI：
10.1371/journal.pone.0089395
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Willey JC
中科院分区：
文献类型：
--
作者：
Yeo J;Crawford EL;Blomquist TM;Stanoszek LM;Dannemiller RE;Zyrek J;De Las Casas LE;Khuder SA;Willey JC
逆转录定量实时PCR（RT-qPCR）检测通过更具临床意义的诊断支持个性化癌症治疗。然而，通过标准临床病理学程序获得的样品是福尔马林固定、石蜡包埋（FFPE）的，并且产生含有PCR干扰物质的低完整性RNA的小样品。需要能够评估FFPE样品的RT-qPCR检测，并具有质量控制和实验室间重现性。我们开发了一种RT-qPCR方法，通过该方法1）相对于已知数量的其各自的竞争性内标分子测量每个基因以控制干扰物质，2）双色荧光水解探针使得能够在实时平台上进行分析，3）控制探针荧光强度变化的外标物，以及4）预扩增来自FFPE RNA样品的最大化信号。针对先前报道的肺癌诊断测试（LCDT）所包含的四个基因开发试剂，然后使用合成天然模板作为测试物品进行分析验证，以评估线性、信号-分析物响应、较低检测阈值、不精密度和准确度。在FFPE正常（N = 10）和恶性（N = 10）肺样本中评估了该方法和这些试剂用于临床检测的适用性。 LCDT所包含的MYC、E2 F1、CDKN 1A和ACTB四种基因中的每一种的试剂具有可接受的线性（R2>0.99）、信号-分析物响应（斜率1.0±0.05）、较低的检测阈值（<10个分子）和不精确性（CV <20%）。泊松分析证实了内标物浓度的准确性。内标物控制实验引入的干扰，防止假阴性，并启用预放大以增加信号而不改变测量值。在使用手术FFPE样本进行双色荧光LCDT目的测试的适用性中，诊断准确率为93%，与先前报告的新鲜样本分析结果相似。这种质量受控的双色荧光RT-qPCR方法将有助于在FFPE临床样本中开发可靠、稳健的基于RT-qPCR的分子诊断检测。
Reverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed. We developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples. Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples. This quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.
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影响因子：
9.3
作者：
Overdevest JB;Theodorescu D;Lee JK
通讯作者：
Lee JK
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
9.4
作者：
Caliendo, Angela M.;Valsamakis, Alexander;Lurain, Nell S.
通讯作者：
Lurain, Nell S.
影响因子：
46.9
作者：
Canales, Roger D.;Luo, Yuling;Goodsaid, Federico M.
通讯作者：
Goodsaid, Federico M.
DOI：
10.1016/s0006-291x(02)00243-7
发表时间：
2002-04-26
影响因子：
3.1
作者：
Crawford, EL;Warner, KA;Willey, JC
通讯作者：
Willey, JC