Comparison of Synthetic, Biotinylated DNA, LNA and PNA Probes for In Situ Hybridization (ISH) Using a Pan-Fungal rRNA Sequence: A Multispectral Imaging Study
KT Montone, MD Feldman. University of Pennsylvania, Philadelphia, PA
Background: ISH for rRNA using DNA oligonucleotide probes can speciate fungal organisms in tissues. Locked nucleic acid (LNA) and peptide nucleic acid (PNA) probes canalso be used for rRNA ISH. LNA are modified RNA nucleotides that exhibit increased thermal stability. PNA is an artificially constructed nucleotide similar to DNA and RNA with stronger binding chanracteristics. A comparsion of these three probe types using multispectral image (MSI) analysis is undertaken in this study.
Design: 7 tissue samples with filamentous fungi were used. DNA, LNA and PNA probes targeting a pan-fungal rRNA sequence were commercially synthesized and 3' biotin-labeled. All probes were utilized at a final concentration of 1 ug/ml. ISH was performed using modified capillary action technology. Tissues were dewaxed, rehydrated and digested with pepsin. Hybridization was for 1 hour at 37oC for DNA and 50oC for LNA/PNA probes. Tissues were washed with either 2XSSC (low stringency) or 0.5 x SSC (high stringency). Hybrids were detected using streptavidin AP and NBT. Slides were examined using a microscope equipped with planapochromatic lenses. Pictures of the fungi using the different probes were obtained at 200 through a liquid crystal filter using a commercially available multispectral imaging system.
Results: All 3 probes identified fungal organisms in all cases. The LNA probe consistently produced the strongest signal under low and high stringency conditions. The LNA probe also had the most background staining but this was reduced following stringent washing. More organisms stained with the LNA probe. By MSI, the average signal intensity was highest for the LNA probe (0.78 (high stringency/0.72 low stringency) followed by the DNA probe (0.44 high stringency/0.37 low stringency) and lastly the PNA probe (0.37 for high and low stringency ). The LNA probe produced a signal which was 1.7-1.9X the DNA probe and 1.8-2.1 times the PNA probe depending on the stringency. The DNA probe signal was found to be 1-1.2X the signal generated with the PNA probe.
Conclusions: DNA, LNA and PNA probes can identify pan-fungal rRNA sequences in tissues. Under the hybridization and washing conditions used in this study, the LNA probe resulted in a 2-fold level of intensity over PNA probe. Even though signal was weaker, in terms of the clarity of staining, the DNA probe compared well to the LNA probe and may be an alternative to LNA probes when one is using ISH for rRNA sequences in paraffin-tissues.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 225, Wednesday Afternoon