Abstract #3030: Systematic evaluation of RNA quality and cDNA conversion of partially degraded RNA from exfoliated cervical cells collected in PreservCyt media for routine clinical evaluation.
Authors: Bridget Riggs, Mujiba Ansari, Abby Koch, Sean Yoder, Steven Enkemann, Erin Siegel
Journal: Cancer Research (2009)

Background: Liquid-based cytological specimens contain exfoliated endo/exo cervical cells collected as part of routine ThinPrep Pap screening. Residual ThinPrep specimens are a tremendous resource of clinical material that can be leveraged for additional biomarker assessment. RNA from exfoliated cells in PreservCyt has been shown to be partially degraded compared to than fresh frozen tissue, which may be due to the nature of differentiated keratinized cells or the preservative media. While RNA has been utilized from PreservCyt media under ideal storage conditions, there are no data on the RNA from samples collected for routine clinical care and using kits specifically for degraded RNA, thus resulting in improved quality of MicroArray chip hybridization. In collaboration with Laboratory Corporation of America Holdings (LabCorp) in Tampa, Fl, we conducted a systematic evaluation of cDNA conversion of ThinPrep RNA using kits designed for degraded RNA, specifically WT-Ovation\#8482; FFPE system V2, (Nugen Technologies, Inc., San Carlos, CA); MessageAmp\#8482; Premier RNA amplification kit, (Applied Biosystems, Foster City, CA) and cDNA-mediated Annealing, Selection, Extension, and Ligation (DASL) Assay® (Illumina, San Diego, CA). Methods: RNA was extracted from 4-mls using Qiagen RNAeasy kit and RNA quality assessed by Agilent Bioanalyzer Picochip. Evaluation was conducted on 1) cDNA conversion efficiency, 2) level of b-actin and 18s rRNA using quantitative RT-PCR, and 3) the percent hybridization and scaling factor assessments from the Microarray hybridization. Samples tested included cervical cell lines; exfoliated cervical cells collected in PreservCyt and process within 1 hour, and ThinPrep samples collected for clinical care received from LabCorp. Results: Overall, RNA from ThinPrep specimens was degraded and the extent of the degradation was not correlated to cytology diagnosis, HPV status, time since collection or total amount of RNA isolated. Based on our preliminary data of two systems, the cDNA conversion of RNA extracted from ThinPreps is poor and resulted in low percent hybridization. However, targeted RT-PCR assessment demonstrated that relative expression levels of a targeted gene, compared to internal controls, can be generated from LabCorp RNA samples. Conclusion: These data support previous finding that RNA from ThinPrep specimens is similar to degraded RNA for other preserved tissues, such as formalin-fixed paraffin embedded tissues. However, unlike FFPE specimens, RNA isolated from ThinPrep specimens does not appear to be optimal for Microarray analyses. Thus DNA-based, not RNA-based, molecular signatures should be identified from clinically relevant and attainable specimens, such as liquid based cytological specimens, to translate functional genomics work from the laboratory to the clinic.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3030.