Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, Ann Arbor, Michigan 48109-0362, USA.
J Mol Diagn. 2010 Sep;12(5):566-75. doi: 10.2353/jmoldx.2010.090107. Epub 2010 Aug 13.
Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression.
Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft.
RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance.
The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens "informative" for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases.
由于缺乏适合可靠定量 RT-PCR（qRT-PCR）的样本，生物标志物在胰腺癌诊断中的应用受到了限制。本研究旨在通过研究胰腺肿块的细针抽吸物（FNA）来确定 RNA 降解的潜在原因，并开发用于准确测量基因表达的方法。
对 32 名患者的样本进行了研究。通过使用甘油醛-3-磷酸脱氢酶的多重 PCR 分析来评估 RNA 降解，通过使用 150-bp 和 80-bp RPS6 扩增子来确定 qRT-PCR 的影响。通过使用胰腺癌异种移植的 FNA 研究了 RNA 降解的潜在原因和规避方法。
从胰腺肿块 FNA 中提取的 RNA 严重降解。片段化与针管直径有关，无法通过改变抽吸技术来克服。甘油醛-3-磷酸脱氢酶的多重 PCR 可区分适合 qRT-PCR 的样本。使用短的 PCR 扩增子（<100bp）可从 FNA 中进行可靠的基因表达分析。当使用适当的样本时，该测定对于基因拷贝数具有高度可重复性，方差最小（0.0003 或约总方差的 0.7%）。
内镜 FNA 的降解特性显著影响基因表达测量的准确性。我们指定用于 qRT-PCR 的“信息性”样本的新方法允许对胰腺疾病的诊断进行准确的分子评估。
