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.
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.
Murphy
Patricia G
PG
BD Diagnostics - TriPath, Durham, NC, USA. patricia_murphy@bd.com
Henderson
Dorian T
DT
Adams
Melissa D
MD
Horlick
Elizabeth A
EA
Dixon
Eric P
EP
King
Lorraine M
LM
Avissar
Patricia L
PL
Brown
Charlotte A
CA
Fischer
Timothy J
TJ
Malinowski
Douglas P
DP
D002584:Cervix Uteri / Q000821:virology*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D005814:Genes, Viral / Q000235:genetics*; D005838:Genotype; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000235:genetics*; D052161:Human papillomavirus 18 / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011309:Preservation, Biological; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D011897:Random Allocation; D011933:Reagent Kits, Diagnostic; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014626:Vaginal Smears
D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012097:Repressor Proteins