An improved RT-qPCR method for quantitation of fragmented mRNAs

用于定量 mRNA 片段的改进 RT-qPCR 方法

• 批准号: 9048312
• 负责人: SERGEI A KAZAKOV
• 金额: $ 28.78万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9048312
• 关键词: Affect; Age; Applications Grants; Archives; Area; Biological Assay; Biological Markers; Breast; Cancer Diagnostics; Cancer Patient; Cell Count; Classification; Cloning; Data; Detection; Development; Diagnosis; Disease; ERBB2 gene; Formalin; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Goals; Gold; Human; Life; Malignant Neoplasms; Malignant neoplasm of prostate; Mammary Gland Parenchyma; Measures; Messenger RNA; Methods; Modeling; Modification; Molecular Profiling; Noise; Oligonucleotide Probes; POLR2A gene; Paraffin Embedding; Phase; Protocols documentation; RNA; Reproducibility; Research; Research Personnel; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Running; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Source; Specificity; Specimen; TFRC gene; Technology; Tissue Sample; Tissues; Validation; Woman; Work; base; breast cancer diagnosis; cancer biomarkers; cancer therapy; commercialization; cost; design; diagnostic assay; histological specimens; improved; mRNA Expression; malignant breast neoplasm; neoplastic cell; new technology; novel; outcome forecast; prognostic; prognostic assays; prototype; public health relevance; transcriptome; transcriptome sequencing; tumor

 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.

 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癌症生物标志物的发现和验证、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，因此目前首选它们用于 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（例如微阵列和测序 (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 碎片的限制，并限制了这些测定的灵敏度和重现性。为了克服这个问题，我们提出了一种检测 FFPE 样品中 mRNA 片段的新方法，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，它可以处理 22-24 nt 的非常短的 mRNA 片段，并在定量 FFPE 样品中的两种模型 mRNA 方面表现出优于 TaqMan RT-qPCR 方法的灵敏度。我们分析了从 8 个乳腺癌和 2 个前列腺癌 FFPE 样本中分离的总 RNA 中的 HER-2（乳腺癌生物标志物）和 GAPDH（内参）mRNA。在第一阶段，我们计划：（i）使用更多的目标 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 能够可靠地定量 FFPE 样品中的 mRNA，其中含有高度片段化的 mRNA，而目前领先的 RT-qPCR 方法无法检测到这些片段； (iv) 确定 mR-FQ 可检测的最大 mRNA 碎片水平。在第二阶段，我们将通过为 20-30 个 BC 生物标志物候选物设计 mRNA-FQ 测定法来走向商业化，并在 80-100 个具有广泛 RNA 碎片水平的 FFPE 样品上验证它们，重点是当前不可用的样品 - 那些含有高度碎片化的 RNA 且无法通过标准 RT-qPCR 方法进行测定的样品。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处理高度碎片化的 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中，以开发和验证改进的乳腺癌（以及其他类型）癌症诊断和治疗预后。