| 1. |
- Regev, A, et al.
(författare)
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The Human Cell Atlas
- 2017
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Ingår i: eLife. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 6
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Bell, Thomas J., et al.
(författare)
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PAIR technology : exon-specific RNA binding protein isolation in live cells
- 2011
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Ingår i: Cell-penetrating peptides. - New York : Humana Press. - 9781607619185 - 9781607619192 ; , s. 473-486
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Bokkapitel (refereegranskat)abstract
- RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP–RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557–1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP–RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.
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| 3. |
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| 4. |
- Peritz, Tiina, et al.
(författare)
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Immunoprecipitation of mRNA-protein complexes
- 2006
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 1:2, s. 577-580
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Tidskriftsartikel (refereegranskat)abstract
- Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)–RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.
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| 5. |
- Spaethling, Jennifer M., et al.
(författare)
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Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 18:3, s. 791-803
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Tidskriftsartikel (refereegranskat)abstract
- Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, IncRNAs and pri-miRNAs. We describe cell-type-and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.
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| 6. |
- Zeng, Fanyi, et al.
(författare)
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A protocol for PAIR : PNA-assisted identification of RNA binding proteins in living cells
- 2006
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 1:2, s. 920-927
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Tidskriftsartikel (refereegranskat)abstract
- All aspects of RNA metabolism are regulated by RNA-binding proteins (RBPs) that directly associate with the RNA. Some aspects of RNA biology such as RNA abundance can be readily assessed using standard hybridization technologies. However, identification of RBPs that specifically associate with selected RNAs has been more difficult, particularly when attempting to assess this in live cells. The peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology has recently been developed to overcome this issue. The PAIR technology uses a cell membrane–penetrating peptide (CPP) to efficiently deliver into the cell its linked PNA oligomer that complements the target mRNA sequence. The PNA will then anneal to its target mRNA in the living cell, and then covalently couple to the mRNA-RBP complexes subsequent to an ultraviolet (UV) cross-linking step. The resulting PNA-RNA-RBP complex can be isolated using sense oligonucleotide magnetic beads, and the RBPs can then be identified by mass spectrometry (MS). This procedure can usually be completed within 3 d. The use of the PAIR procedure promises to provide insight into the dynamics of RNA processing, transport, degradation and translation in live cells.
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