. Global RNA sequencing technologies have revolutionized exploration of gene regulation in health and disease. However, RNA as a molecule is highly susceptible to degradation. As a result, RNA-sequencing, which has become routine in basic research, does not work well with patient specimens because their quality is variable and can not be always controlled or predicted. To improve suitability of patient specimens for transcriptome profiling, an approach for global analysis of RNA polymerase II derived short RNAs will be developed that, unlike existing methods, relies on RNA degradation and works better for low-quality RNA. The approach is based on enzymatic isolation and global sequencing of short 5?-capped RNA (scRNA) fragments and is designed to work with extremely low quality RNA degraded to fragments as short as twenty- five nucleotides. Such low-quality samples are currently impossible to profile with any existing methods.
Aim 1 will establish the order of steps that result in most efficient and specific scRNA recovery, and will compare the new procedure against conventional RNA-sequencing.
Aim 2 will determine the extent of sample degradation when the scRNA sequencing approach becomes beneficial over conventional RNA sequencing. To test the new technology, profiling of preserved specimens previously collected from lung cancer patients will be done and their gene expression profiles will be tested against histological classification previously performed by a pathologist. The work will establish seamless profiling of RNA in cancer specimens across the entire RNA quality range. The procedure is expected to be sufficiently simple for any laboratory experienced in handling next-generation sequencing libraries. This new capability will enable laboratories and sequencing core facilities to accept highly degraded samples and perform comparison of global transcriptomes among degraded and intact specimens. Broader application of the technology will advance collaboration across disciplines including the presently tenuous connection between basic and clinical investigators. ! !
. The novel approach will enable analysis of RNA in cancer specimens whose quality is too low for currently available methods. Improved reliability of RNA profiling will accelerate research on cancer specimens by enhancing collaboration between basic and clinical scientists. Making highly degraded specimens suitable for analysis can help patients avoid unnecessary repeated biopsies and potentially save lives by speeding up diagnosis outcomes.!
