An improved RT-qPCR method for quantitation of fragmented mRNAs

 描述（由申请人提供）：福尔马林固定石蜡包埋（FFPE）组织样本中基因表达的定量对于发现和验证癌症生物标志物、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检测非常灵敏且具有序列特异性，因此目前首选其用于FFPE组织中的mRNA表达谱分析以及验证通过其他表达谱分析方法（如微阵列和测序（RNA-seq））获得的数据。然而，使用这些方法分析FFPE样品受到这些样品中发生的RNA片段化的限制，并限制了这些测定的灵敏度和再现性。为了克服这个问题，我们提出了一种新的方法来测定FFPE样品中的mRNA片段，称为mR-FQ（mRNA片段定量）。在初步研究中，我们开发了一种mR-FQ原型，其可以与22-24 nt的非常短的mRNA片段一起工作，并且在定量来自FFPE样品的两种模型mRNA时证明了其比TaqMan RT-qPCR方法更上级的灵敏度。我们分析了从8个乳腺癌和2个前列腺癌FFPE样品中分离的总RNA中的HER-2（乳腺癌生物标志物）和GAPDH（内部参考）mRNA。在第一阶段，我们计划：（i）使用更多的靶mRNA（5）和更多的FFPE样本（20）验证mR-FQ方法;（ii）进一步优化mR-FQ;（iii）证明mR-FQ可靠地定量FFPE样本中的mRNA，该样本含有高度片段化的mRNA，这些mRNA无法通过目前领先的平行运行的RT-qPCR方法检测;（iv）确定通过mR-FQ可检测的最大mRNA片段化水平。在第二阶段，我们将通过设计用于20-30个BC生物标志物候选物的mR-FQ检测试剂盒，并在80-100个具有广泛RNA片段化水平的FFPE样本上对其进行验证，重点关注目前无法使用的样本-那些含有无法通过标准RT-qPCR方法测定的高度片段化RNA的样本。将比较mR-FQ和RNA-seq分析，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用于高度片段化的RNA。这将使研究人员能够将更广泛的FFPE样本纳入回顾性研究，以开发和验证改进的乳腺癌（以及其他类型的）诊断和治疗方法。

 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.