>> P-493: Comparison of Manual and Automated Methods for Isolation of RNA from BD SurePath(TM) Liquid-Based Pap Specimens
19:00 PM - 19:00 PM
1BD Diagnsotics, Women's Health and Cancer, Durham, NC, USA.
Objective: Progression from high-risk HPV infection to cervical cancer requires dysregulated expression of the HPV E6/E7 oncogenes. Thus, the detection of E6/E7 mRNA may provide useful adjunctive information in equivocal cytology cases. We recently demonstrated that RNA isolated from BD SurePath(TM) liquid-based Pap specimens, using a variety of commercially available manual RNA isolation methods, is an acceptable template for detection of oncogenic HPV transcripts using both real-time RT-PCR and NASBA-based assays. In this study we tested whether RNA prepared from BD SurePath(TM) specimens using a magnetic bead-based method would likewise be suitable for molecular testing.
Methods: Total RNA was isolated from the same BD SurePath(TM) specimens using the manual Ambion RecoverAll(TM) Total Nucleic Acid Isolation kit (glass-fiber filter) and a RecoverAll(TM)-MagMAX(TM) Custom Kit (paramagnetic beads) according to the manufacturer's specifications. The RecoverAll(TM)-MagMAX(TM) kit was utilized in a manual format and also on the automated KingFisher® Particle Processor (Thermo Scientific). Suitability of the RNA for molecular testing was assessed by RT-PCR.
Results: RNA prepared using both the RecoverAll(TM) and RecoverAll(TM)-MagMAX(TM) kits was suitable for use in a real-time RT-PCR application. mRNA from the housekeeping gene GUSB was specifically amplified (CT<38) in 30/30 (100%) specimens isolated using the RecoverAll(TM) kit, 6/7 (86%) samples purified with the manual RecoverAll(TM)-MagMAX(TM) method, and in 22/23 (96%) of RNA preparations isolated using the automated RecoverAll(TM)-MagMAX(TM) method. Samples processed on the automated platform exhibited a 2-10 fold increase in total RNA yield relative to their counterparts isolated using the RecoverAll(TM) kit.
Conclusion: These research data demonstrate that RNA suitable for adjunctive molecular testing, specifically RT-PCR, can be isolated from BD SurePath(TM) liquid-based Pap specimens using the RecoverAll(TM) kit and the RecoverAll(TM)-MagMAX Custom Kit, in either a manual or automated format. Amplification of HPV viral transcripts from these RNA samples is currently ongoing.