Molecular diagnostic tests comprising transcript abundance (TA) measurement have great potential to better predict cancer risk, prognosis, and the optimal therapeutic for each individual. However, this potential is limited by the status of clinical sample collection and processing methods. Methods typically practiced today yield clinical samples with wide variation in RNA quality. Efforts to improve these methods are limited in part by inadequate means to measure RNA quality. Further, use of existing banks of biospecimens with variable RNA quality is limited due to inadequate RNA quality control (QC) measurement methods. This prevents establishment of appropriate cut-off criteria by which to determine which samples will yield reliable reverse transcriptase polymerase chain reaction (RT-PCR) results. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances or methods that either a) interfere with RT efficiency, and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. In pilot studies, quantification of ACTB cDNA molecules/ng RNA controlled for RNA degradation over wider scale than that measured by electropherogram, quantification of CC10 gene gDNA in RNA controlled for gDNA contamination, and measurement of transcript abundance of each gene relative to known number of respective internal standard (IS) molecules controlled for PCR inhibitors. In this proposed project, more comprehensive investigation for the best tests for RNA integrity will be developed and these will be implemented, along with the tests for gDNA contamination and RT inhibition on the novel Standardized NanoArray PCR (SNAP) platform. These methods then will be applied to guide development of improved methods for biospecimen collection.
The specific aims are:
Aim 1. Establish robust tests to measure RNA integrity, gDNA contamination, and RT inhibition along with accompanying reference materials Aim 2. Use the robust RNA QC methods to establish cut-off thresholds for reliable measurement of transcript abundance based diagnostic tests by RT-PCR or microarray and guide improvements in methods for collecting and processing clinical biospecimens.
Aim 3. Demonstrate the feasibility of implementing the RNA QC tests either alone or together with disease specific assays on commercial platforms. Successful completion of the proposed project will enable identification of tissue sample collection and processing methods that are more likely to yield RNA samples suitable for molecular diagnostic studies. Further, it will enable more accurate determination of which clinical biospecimens collected according to present methods and/or in existing tissue banks will yield reliable results for promising transcript abundance based diagnostic tests.
Sequencing of the human genome has opened opportunities for advances in diagnostics based on transcript abundance measurement. This promise has been hampered by lack of adequate tests for RNA quality. Development of improved RNA Quality Control tests will enable development of improved biospecimen collection methods and improved methods to determine which RNA samples have sufficient quality to yield reliable transcript abundance values. These advances will lead to more reliable testing required for personalized medicine in the field of cancer diagnosis and management.
| Blomquist, Thomas; Crawford, Erin L; Yeo, Jiyoun et al. (2015) Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing. Biomol Detect Quantif 5:30-37 |
| Yeo, Jiyoun; Crawford, Erin L; Blomquist, Thomas M et al. (2014) A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples. PLoS One 9:e89395 |
| Stanoszek, Lauren M; Crawford, Erin L; Blomquist, Thomas M et al. (2013) Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples. J Mol Diagn 15:391-400 |
| Blomquist, Thomas M; Crawford, Erin L; Lovett, Jennie L et al. (2013) Targeted RNA-sequencing with competitive multiplex-PCR amplicon libraries. PLoS One 8:e79120 |
