Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath™ preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR

Abstract

This study was performed to demonstrate that RNA isolated from cell lines and cervical cytology specimens stored in SurePath™ preservative fluid would be functional in real-time RT-PCR assays.RNA was isolated from cervical cell lines or cytology samples stored in SurePath™ preservative at room temperature for 2–5 weeks using five commercially available RNA purification kits, three of which contain proteinases. The quality of the RNA was assessed by real time RT-PCR amplification of GAPDH, GUSB, U1A, HPV 16 and 18 E6 mRNAs.RNA was isolated successfully from cells that were stored in SurePath™ preservative fluid with only the three protocols that contained proteinases. GAPDH was amplified in 98–100% of the samples, GUSB in 90–98%, and the least abundant transcript, U1A, was amplified in 81–96% of the samples. HPV 16 and 18 E6 transcripts were detected in 56% of high grade, 39% of low grade and 2% of normal samples, with a concordance between DNA genotype and E6 mRNA expression of 97%.We demonstrated that RNA can be extracted from cervical cell lines and cytology specimens stored in BD SurePath™ preservative fluid with three different procedures that all contain proteinases. This RNA is suitable for real-time RT-PCR applications.