A method of simultaneously detecting target nucleic acids and target proteins in a biological sample, comprising treating the biological sample with a crosslinking agent, that is after incubating it with a primary antibody that detects the target proteins and prior to detecting the target nucleic acids by in situ hybridization.

A method of simultaneously detecting target nucleic acids and target proteins in a biological sample, comprising treating the biological sample with a crosslinking agent, that is after incubating it with a primary antibody that detects the target proteins and prior to detecting the target nucleic acids by in situ hybridization.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

Methods, kits, and systems for fixation of RNA permitting its detection in intact tissue, such as, large volume of mammalian tissue are disclosed. The methods, kits, and systems utilize carbodiimide-based chemistry to stably retain RNAs in tissue clarified using CLARITY. Also provided herein are methods, kits, and systems for detection of RNAs in clarified tissue.

Methods, kits, and systems for fixation of RNA permitting its detection in intact tissue, such as, large volume of mammalian tissue are disclosed. The methods, kits, and systems utilize carbodiimide-based chemistry to stably retain RNAs in tissue clarified using CLARITY. Also provided herein are methods, kits, and systems for detection of RNAs in clarified tissue.

In certain embodiments, this application discloses methods for detecting lung cancer. The method includes characterization of cells extracted from human sputum, which is a valuable tissue surrogate and source of upper respiratory cells that become cancerous early in 5 the process of lung cancer development. The method includes the staining of extracted cells with fluorescent reporters that produce a specific pattern in the nuclei of labeled cells, which can be made visible by light microscopy. The pattern is relevant to a type of epigenetic coding of DNA known as DNA methylation, which changes in specific cells of the lung during cancer development, in comparison to normal respiratory cells.

In certain embodiments, this application discloses methods for detecting lung cancer. The method includes characterization of cells extracted from human sputum, which is a valuable tissue surrogate and source of upper respiratory cells that become cancerous early in 5 the process of lung cancer development. The method includes the staining of extracted cells with fluorescent reporters that produce a specific pattern in the nuclei of labeled cells, which can be made visible by light microscopy. The pattern is relevant to a type of epigenetic coding of DNA known as DNA methylation, which changes in specific cells of the lung during cancer development, in comparison to normal respiratory cells.

Quantification of RNAs is one of the most essential tools to characterize cells. This tool is widely used in disease diagnosis, pharmacogenomics, and drug development. Single cell RNA fluorescent in situ hybridization (smRNA-FISH) revolutionized RNA detection and quantification by detecting every single RNA molecule of a gene. However, this technology is incapable of assaying relatively short RNAs, and it suffers from high cost and low throughput. Here, we describe a technology that simultaneously overcomes the three drawbacks using conventional instrumentation. This QD-smRNA-FISH technology uses hybridization of quantum dot-labeled DNA oligonucleotides to the RNA molecules for visualization and counting. Quantum dots (QDs) have been assumed inapplicable to counting individual RNA molecules, due to the well-known blinking problem (display intermittency) (Medintz I L, Uyeda H T, Goldman E R, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4: 435-446). This problem has been circumvented by this new experimental design. In some embodiments described herein, the methods assemble several QDs to every target RNA molecule and leverage the complementation of the QDs to achieve an overall non-intermittent signal on each target molecule. We validated QD-smRNA-FISH by comparing its signals with those of standard smRNA-FISH. We successfully applied QD-smRNA-FISH to test the interaction of two RNAs, a task that cannot be accomplished with standard smRNA-FISH. The QD-smRNA-FISH method offers a highly accurate method for single RNA molecule detection and counting under standard fluorescent microscopes, and enables analysis of relatively short RNAs (<1000 bases) which comprises the majority of eukaryotic transcriptome and more than half of the eukaryotic mRNAs. The QD-smRNA-FISH method also reduces the reagent cost by several folds and allows for analysis of multiple genes in parallel.

Quantification of RNAs is one of the most essential tools to characterize cells. This tool is widely used in disease diagnosis, pharmacogenomics, and drug development. Single cell RNA fluorescent in situ hybridization (smRNA-FISH) revolutionized RNA detection and quantification by detecting every single RNA molecule of a gene. However, this technology is incapable of assaying relatively short RNAs, and it suffers from high cost and low throughput. Here, we describe a technology that simultaneously overcomes the three drawbacks using conventional instrumentation. This QD-smRNA-FISH technology uses hybridization of quantum dot-labeled DNA oligonucleotides to the RNA molecules for visualization and counting. Quantum dots (QDs) have been assumed inapplicable to counting individual RNA molecules, due to the well-known blinking problem (display intermittency) (Medintz I L, Uyeda H T, Goldman E R, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4: 435-446). This problem has been circumvented by this new experimental design. In some embodiments described herein, the methods assemble several QDs to every target RNA molecule and leverage the complementation of the QDs to achieve an overall non-intermittent signal on each target molecule. We validated QD-smRNA-FISH by comparing its signals with those of standard smRNA-FISH. We successfully applied QD-smRNA-FISH to test the interaction of two RNAs, a task that cannot be accomplished with standard smRNA-FISH. The QD-smRNA-FISH method offers a highly accurate method for single RNA molecule detection and counting under standard fluorescent microscopes, and enables analysis of relatively short RNAs (<1000 bases) which comprises the majority of eukaryotic transcriptome and more than half of the eukaryotic mRNAs. The QD-smRNA-FISH method also reduces the reagent cost by several folds and allows for analysis of multiple genes in parallel.