An improved RT-qPCR method for quantitation of fragmented mRNAs

 描述(申请人提供)：量化福尔马林固定石蜡包埋(FFPE)组织样本中的基因表达对于发现和验证癌症生物标记物、肿瘤分类以及评估癌症治疗过程中的进展非常重要。由于RT-qPCR分析非常敏感和序列特异性，目前它们是FFPE组织中mRNA表达谱以及通过微阵列和测序(RNA-seq)等其他表达谱方法获得的数据验证的首选方法。然而，使用这些方法来分析FFPE样品受到这些样品中发生的RNA碎片的限制，并限制了这些分析的灵敏度和重复性。为了克服这个问题，我们提出了一种新的检测FFPE样品中mRNA片段的方法，称为MR-FQ(mRNA片段量化)。在初步研究中，我们开发了一个MR-FQ原型，它可以处理22-24个核苷酸的很短的mRNA片段，并证明了它在定量FFPE样本中的两个模型mRNA时比TaqMan RT-qPCR方法更灵敏。我们从8例乳腺癌和2例前列腺癌FFPE标本的总RNA中分析了HER-2(乳腺癌生物标志物)和GAPDH(内参)mRNAs。在第一阶段，我们计划：(I)使用更多的靶mRNAs(5个)和更多的FFPE样本(20个)来验证MR-FQ方法；ii)进一步优化MR-FQ；(Iii)证明MR-FQ可靠地定量包含当前领先的RT-qPCR方法无法并行运行的高度片段化的mRNAs的FFPE样本中的mRNAs；(Iv)确定MR-FQ可以检测到的最大mRNA片段水平。在第二阶段，我们将走向商业化，为20-30 BC候选生物标记物设计MR-FQ分析，并在具有广泛RNA片段化水平的80-100个FFPE样本上进行验证，重点是目前无法使用的样本-那些包含标准RT-qPCR方法无法检测的高度片段化RNA的样本。MR-FQ和RNA-SEQ分析将进行比较，我们预计MR-FQ将能够验证RNA-SEQ结果，因为这两种方法都可以处理高度片段的RNA。这将使研究人员能够将更广泛的FFPE样本纳入回溯性研究，以开发和验证改进的乳腺癌(以及其他类型的)癌症诊断和治疗预后。

 DESCRIPTION (provided by applicant): Quantification of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples is important for the discovery and validation of cancer biomarkers, for tumor classification, and to assess progress during cancer treatment. Because RT-qPCR assays are very sensitive and sequence-specific, they are currently preferred for mRNA expression profiling in FFPE tissues as well as for validation of data obtained by other expression profiling methods such as microarrays and sequencing (RNA-seq). However, use of these methods to analyze FFPE samples is constrained by the RNA fragmentation that occurs in these samples and limits the sensitivity and reproducibility of these assays. To overcome this problem, we proposed a novel method for assaying mRNA fragments in FFPE samples, called mR-FQ (mRNA Fragment Quantification). In Preliminary Studies, we developed a mR-FQ prototype, which can work with very short mRNA fragments of 22-24 nt and demonstrated its superior sensitivity over the TaqMan RT-qPCR method in quantifying two model mRNAs from FFPE samples. We analyzed HER-2 (a breast cancer biomarker) and GAPDH (internal reference) mRNAs in total RNA isolated from 8 breast cancer and 2 prostate cancer FFPE samples. In Phase I, we plan to: (i) validate the mR-FQ method using a larger number of target mRNAs (5) and more FFPE samples (20); ii) further optimize mR-FQ; (iii) demonstrate that mR-FQ reliably quantifies mRNAs in FFPE samples containing highly fragmented mRNAs that are not detectable by currently leading RT-qPCR methods run in parallel; (iv) determine the maximum mRNA fragmentation level detectable by mR-FQ. In Phase II, we will move towards commercialization by designing mR-FQ assays for 20-30 BC biomarker candidates and validating them on 80-100 FFPE samples having a wide range of RNA fragmentation levels with focus on currently unusable samples-those containing highly fragmented RNA that cannot be assayed by standard RT-qPCR methods. mR-FQ and RNA-seq analyses will be compared and we expect that mR-FQ will be able to validate RNA-seq results since both methods can work with highly fragmented RNA. This will allow researchers to include a wider range of FFPE samples into retrospective studies for the development and validation of improved breast (as well as other types of) cancer diagnostics and treatment-prognostics.