Visceral Adipose Tissue (VAT) is an active metabolic organ that consists mainly of mature adipocytes and cells of the vascular fraction (SVF), which releases different bioactive molecules that control the process of metabolism, hormones, and immune; At present, it is not clear how this process is regulated in the Adipose Network. Therefore, the development of methods that evaluate the contribution of each cell population to the pathophysiology of adipose tissue is very important.
This protocol describes the isolation measures and provides the problem solving guidelines needed for efficient insulation of proper mature adipocytes and SVF from human VAT biopsy in one process, using the enzymatic digestive technique of collagenase.
In addition, the protocol was also optimized to identify the association of macrophages and insulated mature RNA adipocytes for gene expression studies, which enabled the appearance of research that dissected the interaction between the population of this cell. Briefly, the PPN biopsy is washed, mechanically chopped, and digested to produce a single cell suspension. After centrifugation, the mature adipocyte isolated by flotation from SVF pellets. The RNA extraction protocol ensures high total RNA results (including MIRNAS) from adipocytes for downstream expression tests. Simultaneously, SVF cells are used to characterize the association of macrophages (pro and anti-inflammatory phenotypes) through analysis of the flow of cytometry.
High yield insulation and quality RNA from lung slices cut with human precision for the integration of RNA sequencing and computing with a larger patient cohort
Sliced lungs cut precision (PCL) has increased increasing interest as a model for studying biology / lung disease and novel therapy screening. In particular, PCL originating from human networks can better recapitulate several aspects of biology / lung disease compared to animal models. Some experimental readings have been established for use with PCL, but obtain high yields and quality of RNA for downstream analysis remain challenging. This is very problematic to utilize the power of the next generation sequencing technique, such as Sequencing RNA (RNA-SEQ), for non-biased and high-placed analysis of PCLS human cohorts.
In the current study, we present a new approach to isolate high-quality RNA from a small number of networks, including sick human networks, such as idiopathic lung fibrosis (IPF). We show that RNA isolated using this method has enough quality for analysis of RT-QPCR and RNA-SEQ. Furthermore, RNA-SEQ data from PCLS Human can be used in several set computing pipes, including Data Deconvolution of Bulk RNA-SEQ using single cell RNA-SEQ data available publicly. Dekonvolution uses Bisque reveals the diversity of cell populations on human PCLs, including some immune cell populations, which correlate with the population of cells known to be present and deviate in human diseases.
Single cell isolation of murine muscle regeneration for analysis sequencing RNA
Sequencing a single cell RNA (SCRA-SEQ) is a strong technique for deconvoluting and grouping thousands of cells mixed based on their gene expression. Here, we present a complete protocol for an impartial evaluation to regenerate the murine framework muscle using SCRNA-SEQ. The skeleton muscles are unique in their cellular composition as multinucleused muscle cells (myofibers). The protocol focuses on mononuclear cell isolation from the muscles for the next SCNA-SEQ analysis and can be modified to assess cell populations on other interesting networks. For complete details about the use and implementation of this protocol, please refer to Liu et al. (2015) and OPRESCU et al. (2020).
Methods for simultaneous and quantitative insulation of mitochondrial DNA, nuclear DNA and RNA from mammalian cells
The purpose of this study was to assess two protocols for their capacity to simultaneously isolate RNA, MTDNA and NCDNA from mammalian cells. We compared Trizol Invitrogen-based methods and Qiengen Dneeny columns, using HEPG2 cells and Human Primary Human Globlastoma stem cells. Both methods allow isolation from the three types of nucleic acids and provide similar results in MTDNA. However, the results in NCDNA are more than tenfold in the column, as observed for both cell types. Conversely, the Trizol method proved to be more reproductive and is the method of choice to isolate the RNA from glioblastoma cells, as shown for the housekeeping gene RPLP0 and RPS9.
Nucleus isolation root plants for single cell RNA sequencing
Characterization of the similarity of transcription and differences between plant cells and cell types is important to better understand the biology of each cell that composes the factory, to disclose a new molecular mechanism that controls gene activity, and ultimately applies a meaningful strategy to increase plant biology. To get a deeper understanding of regulations on plant gene activity, individual transcription of each plant cell needs to be established. Until now, most single cell approach is limited to mass transcriptom studies on selected cell types.
Accessing certain cells is needed to develop labor-intensive strategies. Recently, a single cell sequencing strategy was successfully applied to the isolated arabidopsis root protoplasts. However, this strategy depends on successful insulation on proper protoplasts on the optimization of enzymatic cocktails needed to digest cell walls and in the compatibility of fragile plant protoplasts using the microfluidal system to produce a single cell transkriptom library. To overcome these difficulties, we present a simple and fast alternative strategy: insulation and use of the core of the plant to access significant transcriptom information from plant cells. This protocol is specifically developed to allow the use of nuclei plants with the technology of the ‘genomics’ chromium technology partition. Briefly, the nuclei plant is released from the root by cutting the nuclei insulation buffer before purifying with nuclus filtration.
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After sorting, the nuclei was referred to in a low divalent ion buffer compatible with chromium technology to make a single ribonucleic acid sorting library (Snucrna-seq). © 2020 Wiley Peripics LLC. Basic Protocol 1: Arabidopsis seed sterilization and basic planting 2: nuclei insulation from Arabidopsis Basic Protocol Roots (fans) Sort Nuklak (Fans) Protocol: Nuclei Density Estimates Using Countess II Cell Counter Alternate Protocol 1: Growth Conditions for Medicago Truncatula and Sorghum Bicolor Alternate Protocol 2: The estimated nuclei density uses Snucrna-SEQ technology.