|
Epicentre Forum 6 (2)
||
M. O'Connell, M. Cohenford, and B. Lentrichia, Cytyc Corporation,
Boxborough, MA
Anatomic molecular pathologists often rely on preserved tissue samples
rather than cytological specimens to perform molecular assays on archived
samples. This article describes a system that combines a method for collecting
and preserving cytological samples with an easy-to-use technology for
isolating PCR amplifiable products.
The ThinPrep® System (Cytyc Corp.) of sample collection and slide
preparation is designed to improve the appearance and diagnostic accuracy
of cytology slides. PreservCyt® Solution (Cytyc Corp.), used with
the ThinPrep System, is a methanol-based collection medium that preserves
the morphologic integrity of cervical cells during transportation and
storage. PreservCyt Solution also preserves cellular proteins and nucleic
acids within gynecological samples. Once the ThinPrep slide is made,
remaining specimen can be used for further analysis such as Human Papillomavirus
(HPV) testing. Applications such as in situ hybridization and
immunocytochemistry have been previously performed on these types of
samples,1-4 but procedures for extracting DNA from cells suspended
in PreservCyt Solution for polymerase chain reaction (PCR) have not been
well defined. Conventional methods for extracting DNA are often time-consuming
and rely on the use of caustic reagents. Moreover, some of these procedures
produce low yields of PCR-quality DNA.
Here, we describe a research application using the MasterAmp™ DNA
Extraction Solution (Epicentre). The MasterAmp Solution was used to successfully
purify HPV DNA from cervical cells collected into PreservCyt Solution
yielding DNA free of PCR inhibitors, as demonstrated by amplification
of a region of the HPV gene. Because the entire extraction reaction was
performed in one tube, DNA yield was not compromised. beta-globin
primers were used as internal controls to determine the integrity of
the PCR reagents.5 Cell cultures positive and negative for
HPV DNA were used as additional controls.
Samples
Residual exfoliated cervical cells collected into PreservCyt Solution
and processed for the ThinPrep Processor were used in this study. Positive
control (CaSki cells, 600 viral copies of HPV DNA/cell) and negative
control (C-33A HPV negative cells) culture cell samples were collected
and stored in PreservCyt Solution. All control samples were processed
in an identical manner to the clinical samples as described below.
DNA extraction
DNA extraction was performed using the MasteAmp DNA Extraction Solution
as follows: 1) 1-2 ml of PreservCyt sample was transferred to Eppendorf
microcentrifuge tubes and centrifuged at 13,000 rpm for 5 minutes. The
supernatants were removed and the cell pellets were resuspended in 500 µl
of MasterAmp DNA Extraction Solution; 2) caps were screwed tightly and
specimens were mixed by vortexing for 15 seconds. The tubes were then
incubated at 60°C for 30 minutes; 3) the tubes were vortex mixed for
15 seconds and transferred to a 98°C waterbath for 8 minutes, mixed again,
and transferred back to the 98°C waterbath for an additional 8 minutes.
Then, the samples were mixed by vortexing and chilled on ice; 4) cellular
debris was removed by centrifugation at 13,000 rpm for 5 minutes and
supernatants containing purified DNA were transferred to a clean Eppendorf
tube. DNA was stored at -20°C prior to analysis.
PCR amplification and detection
PCR conditions were as follows: 1.0 µM of each HPV primer (MY09/MY11,
Research Genetics) and 0.1 µM of each ß-globin primer (GH20bg1/GH20bg2,
Genosys Biotechnologies) were added to a reaction containing 2.5 units
of GeneAmp™ AmpliTaq™ DNA Polymerase (Perkin Elmer), 200 µM
of each dNTP (Perkin Elmer), 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM
MgCl2, and dH2O to a total volume of 50 µl. Lambda
DNA and lambda primers were used as internal reagent controls following
the same protocol as described above. The PCR conditions were 95°C for
4 minutes; followed by 35 cycles of 95°C for 1 minute, 55°C for 1 minute,
and 72°C for 1 minute; followed by a final extension at 72°C for 10 minutes.
PCR amplicons were detected by 1% agarose gel electrophoresis at 75 volts
for 45 minutes. Bands were visualized under UV light using EtBr stain.
As shown in Figure 1, the ß-globin gene was amplified from cervical
cell or tissue culture cell DNA extracted using the MasterAmp DNA Extraction
Solution (Lanes 3-6). HPV DNA could also be amplified from the virus-positive
cell line CaSki (Lane 6), but was not amplified from the HPV negative
cell line C-33A (Lane 5). Results also reveal the amplification of HPV
DNA from HPV-positive cervical samples stored in PreservCyt Solution
(Lane 4). As expected, no PCR product was obtained from the HPV-negative
samples (Lane 3).
 |
Figure 1. Representative PCR amplifications. PCR was performed
as described in the text. Lane 1, dH2O negative control; Lane 2,
lambda positive control; Lanes 3 & 4, cervical samples; Lane 5, HPV
negative control cell line C-33A; Lane 6, HPV positive cell line
CaSki. |
Table 1 summarizes the results of the 30 cervical samples that were
PCR amplified to detect the presence of both the ß-globin gene and HPV
DNA. Thirty of 30 samples were positive for the ß-globin gene indicating
that successful PCR amplification was achieved using DNA isolated with
the MasterAmp DNA Extraction Solution.
| HPV |
30 |
14 |
| β - globin |
0 |
30 |
The Epicentre MasterAmp DNA Extraction Solution was used to successfully
isolate DNA from cervical cells collected into PreservCyt Solution. The
HPV DNA also appeared to be free of any PCR inhibitors as it was readily
amplified by Taq DNA polymerase indicating the suitability of
MasterAmp DNA Extraction Solution for isolating PCR-ready DNA. Therefore,
PCR amplification can be used as a research application tool for the
detection of HPV DNA from cellular material in PreservCyt Solution.
- Kurtycz, D. et al. (1996) Diagnostic Cytopathology 15
(1), 46.
- Lentrichia, B. et al. (1994) 83rd Annual Meeting
USCAP.
- Hampton, R.W. et al. (1996) Am. J. Pathol. 149, 1782.
- Ting, Y. and Manos, M.M. (1990) in: PCR Protocols: A Guide to
Methods and Application, ed. Innis, M.A. et al., Academic Press,
Inc., CA, 356.
- Bauer, H. et al. (1991) JAMA 265, 472.
- Saike, R. et al. (1985) Science 230, 1350.
|
