Abstract 825: Development of RNA quality-control methods to improve tissue archiving and molecular diagnostic testing

Abstract

Abstract Biospecimen collection and processing methods practiced today yield clinical samples with a wide variation in RNA quality. Efforts to improve these methods are inadequate due to insufficient means to measure RNA quality. Improved RNA quality control (QC) measurement methods will enable establishment of cut-off criteria to determine which biospecimens will yield reliable RT-PCR results. Implementation of such criteria will enable existing banks of samples with variable RNA quality to be used for reverse transcriptase polymerase chain reaction (RT-PCR)-based diagnostic test development. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances that either a) interfere with RT efficiency and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. An RNA integrity reference model was established by incubating A549 cell line populations at room temperature for different amounts of time following cytolysis. Between 0 and 100 min. post cytolysis, ACTB degradation, as well as the degradation of other major housekeeping genes, was more rapid than decline in RNA Integrity Number (RIN) Score, a measurement based on microcapillary electropheresis. Quantification of single-copy CC10 gene gDNA in RNA controlled for gDNA contamination and measurement of transcript abundance of each gene relative to known number of respective cDNA internal standards controlled for PCR inhibitors. The RT inhibition test uses a Reverse Transcription Standards Mixture (RTSM) that contains a known number of copies of ERCC (External RNA Control Consortium) RNA and cDNA standards. A known quantity of RTSM is added to an RNA sample and measurement of PCR product from the RNA standard relative to its cDNA standard following RT-PCR assesses RT efficiency. These RT-PCR tests have been implemented on an Agilent 2100 Bioanalyzer® and are being developed for implementation on high throughput RT-PCR platforms such as the Biotrove OpenArray® nanoarray and the Gene Express Standardized Expression Measurement (SEM) Center™. Improved methods for biospecimen collection will be selected and will then be used to identify which samples collected according to present methods are suitable for analysis by RT-PCR-based molecular diagnostic tests. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 825.