Dwaine A. Braasch

Validating Bioluminescence Imaging as a High-Throughput, Quantitative Modality for Assessing Tumor Burden

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

Synthesis and Purification of Peptide Nucleic Acids

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.

Cellular Delivery of Locked Nucleic Acids (LNAs)

Locked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. They are synthesized using standard DNA/RNA synthesis methods, and have a negatively charged backbone that confers good solubility. This unit describes a method for the introduction of LNA oligomers into cells. A support protocol also describes the determination of melting temperatures for LNA oligomers.