NCI-Frederick View Institution's Website 5 articles published in JoVE Cancer Research Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model Robert Y. S. Cheng1, Nimit L. Patel2, Timothy Back1, Debashree Basudhar1, Veena Somasundaram1, Joseph D. Kalen2, David A. Wink1, Lisa A. Ridnour1 1Molecular Mechanism Section, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute at Frederick, 2Small Animal Imaging Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique. Biology SpOT the Correct Tissue Every Time in Multi-tissue Blocks Anna Coffey1,2, Michael D. Johnson3, Deborah L. Berry3 1Center for Advanced Preclinical Research (CAPR), Frederick National Laboratory for Cancer Research, 2Leidos Biomedical Research, Inc., 3Department of Oncology, Georgetown University The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections. Biology RNA Secondary Structure Prediction Using High-throughput SHAPE Sabrina Lusvarghi*1, Joanna Sztuba-Solinska*1, Katarzyna J. Purzycka1, Jason W. Rausch1, Stuart F.J. Le Grice1 1RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution. Biology Isolation of Immune Cells from Primary Tumors Stephanie K. Watkins1, Ziqiang Zhu1, Keith E. Watkins, Arthur A. Hurwitz1 1Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute - Frederick In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue. Immunology and Infection Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays Helene Mens1, Mary Kearney1, Ann Wiegand1, Jonathan Spindler1, Frank Maldarelli1, John W. Mellors2, John M. Coffin3 1The virology Core at the HIV Drug Resistance Program, NCI-Frederick, 2Division of Infectious Diseases, University of Pittsburgh, 3Department of Molecular Biology and Microbiology, Tuffts University Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.