An improved RT-qPCR method for quantitation of fragmented mRNAs



 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.