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

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• 作者：Patricia G. Murphy ; Dorian T. Henderson ; Melissa D. Adams ; Elizabeth A. Horlick ; Eric P. Dixon ; Lorraine M. King ; Patricia L. Avissar ; Charlotte A. Brown ; Timothy J. Fischer ; Douglas P. Malinowski
• 关键词：SurePath™ ; Real-time RT-PCR ; HPV ; RNA ; Liquid-based cytology ; Cervical cancer
• 刊名：Journal of Virological Methods
• 出版年：2009
• 出版时间：March 2009
• 年：2009
• 卷：156
• 期：1-2
• 页码：138-144
• 全文大小：386 K

文摘

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.