In-situ hybridization in PreservCyt solution using PNA probesPublic
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PNA (peptide nucleic acid) is a synthetic nucleic acid analog consisting of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds instead of the sugar-phosphate backbone on DNA (Goforth, 2000). PNA is frequently referred to as a "DNA mimic" as it obeys Watson-Crick base-pairing rules for hybridization to a complementary nucleic acid target (Nielsen and Egholm, 1999). In this project, a PNA probe was designed against the L1 gene of carcinogenic human papilloma viral strain-16 (HPV-16), and tested using in-situ hybridization (ISH) on HPV-infected and unifected cell lines preserved in Cytyc's patented PreservCyt Solution. Initially only a weak fluorescent signal was observed on Ca Ski cells, perhaps because the PreservCyt solution hinders the permeability of cells. In order to improve the signal intensity several pretreatment steps, such as trypsin and pepsin digestion, detergent, and microwave heating were applied before hybridization. The combination of pepsin digestion with sodium citrate buffer rendered the brightest fluorescent signal. Additionally, a ThinPrep? 2000 processor wash step was used to substitute the conventional centrifugation wash step after overnight hybridization. The ThinPrep? 2000 processsor wash step not only reduced the background problem on the slide, but also provided a brighter hybridization signal since all the unbound probe was removed.
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