RESUMO
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Assuntos
Proteínas , RNA , Humanos , Inclusão em Parafina , RNA/genética , Imagem Molecular , FormaldeídoRESUMO
Digital Spatial Profiling (DSP) is a method for highly multiplex spatial profiling of proteins or RNAs suitable for use on formalin-fixed, paraffin-embedded (FFPE) samples. The approach relies on (1) multiplexed readout of proteins or RNAs using oligonucleotide tags; (2) oligonucleotide tags attached to affinity reagents (antibodies or RNA probes) through a photocleavable (PC) linker; and (3) photocleaving light projected onto the tissue sample to release PC oligonucleotides in any spatial pattern across a region of interest (ROI) covering 1 to ~5,000 cells. DSP is capable of single-cell sensitivity within an ROI using the antibody readout, with RNA detection feasible down to ~600 individual mRNA transcripts. We show spatial profiling of up to 44 proteins and 96 genes (928 RNA probes) in lymphoid, colorectal tumor and autoimmune tissues by using the nCounter system and 1,412 genes (4,998 RNA probes) by using next-generation sequencing (NGS). DSP may be used to profile not only proteins and RNAs in biobanked samples but also immune markers in patient samples, with potential prognostic and predictive potential for clinical decision-making.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de RNA , Software , Análise Espacial , Fixação de TecidosRESUMO
We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.
Assuntos
Sondas de DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Linhagem Celular , Cor , Sondas de DNA/genética , Biblioteca Gênica , Genes Reporter , Humanos , Processamento de Imagem Assistida por Computador , Análise de Sequência com Séries de Oligonucleotídeos , Poliovirus , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Thick filaments are generally thought to be effectively inextensible. Here we use novel nanofabricated cantilevers to carry out the first direct force-elongation measurements on single vertebrate thick filaments. Cantilevers are ideal for these experiments: force ranges are from pico- to micronewtons, specimens can be visualized during the experiment, and attachment surfaces are in the same plane as the filament. Synthetic thick filaments from rabbit myosin were suspended between two cantilevers and stretched. With stretch, stiffness increased gradually and then became nearly constant after approximately 100 pN. Stretch rate had little or no effect on force-elongation behavior. Under physiological loads (approximately 240 pN axially averaged with full activation) filaments elongated by 1.1 +/- 0.3%. Previous x-ray diffraction results showed a 1.0 to 1.5% increase in myosin head spacing with activation; however, this increase in spacing has been interpreted as change in the state of the cross-bridges, not as elasticity in the thick filament backbone. Comparison with our data suggests that changes in the myosin x-ray reflections seen during activation may be due to elongation of the thick filament backbone. Recognition of thick filament elasticity is important because it affects the interpretation of mechanical experiments and inferences drawn on the molecular mechanism of contraction.
