ABSTRACT
In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Lymphoma, B-Cell/genetics , Lymphoma, Non-Hodgkin/genetics , Paraffin Embedding/methods , Tissue Fixation/methods , Translocation, Genetic , Computational Biology/methods , Gene Rearrangement , Genes, bcl-2/genetics , Genes, myc/genetics , Humans , In Situ Hybridization, Fluorescence/methods , Lymphoma, B-Cell/diagnosis , Lymphoma, Non-Hodgkin/diagnosis , Proto-Oncogene Proteins c-bcl-6/genetics , Reproducibility of Results , Retrospective Studies , Sensitivity and SpecificityABSTRACT
Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.
Subject(s)
Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Keratinocytes/cytology , Single-Cell Analysis/methods , Cell Differentiation , Cells, Cultured , Gene Expression Profiling/methods , Gene Expression Regulation , Humans , Keratinocytes/chemistry , Phosphorylation , Proteomics/methods , Quinazolines/pharmacology , Tissue Fixation , Tyrphostins/pharmacologyABSTRACT
In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells.
Subject(s)
Hydrogels/chemistry , Nucleic Acid Amplification Techniques , RNA, Messenger/analysis , Actins/genetics , DNA Primers/chemistry , DNA Primers/metabolism , Fluorescent Dyes/chemistry , Humans , K562 Cells , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Proto-Oncogene Proteins c-myc/genetics , Single-Cell AnalysisABSTRACT
We demonstrate a technique for controlling the content of multiple microdroplets in time. We use this system to rapidly and quantiatively determine the solubility diagrams of two model proteins (lysozyme and ribonuclease A).