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Cold Spring Harbor Protocols

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https://www.readbyqxmd.com/read/30510132/mapping-of-in-vivo-rna-binding-sites-by-ultraviolet-uv-cross-linking-immunoprecipitation-clip
#1
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
RNA "CLIP" (cross-linking immunoprecipitation), the method by which RNA-protein complexes are covalently cross-linked and purified and the RNA sequenced, has attracted attention as a powerful means of developing genome-wide maps of direct, functional RNA-protein interaction sites. These maps have been used to identify points of regulation, and they hold promise for understanding the dynamics of RNA regulation in normal cell function and its dysregulation in disease.
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510131/optical-transfection
#2
Priti Kumar, Arvindhan Nagarajan, Pradeep D Uchil
Strategies for the delivery of genes into eukaryotic cells fall into three categories: transfection by biochemical methods, transfection by physical methods, and virus-mediated transduction. "Optical transfection"-a physical transfection method-exploits the ability of light to create small transient pores in the plasma membrane of mammalian cells.
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510130/sampling-and-preparation-of-rabbit-serum
#3
Edward A Greenfield
Periodic test bleeds collected from immunized animals should be checked for the desired antibodies. For rabbits, test bleeds normally are performed from the marginal ear vein. Five to 10 mL can be collected conveniently and will provide more than enough serum for most tests.
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510129/preparing-antigens-using-a-baculovirus-expression-system
#4
Edward A Greenfield, James DeCaprio, Mohan Brahmandam
Baculovirus-produced recombinant proteins circumvent many of the issues that limit bacterial protein production. Baculoviruses contain double-stranded, circular, supercoiled DNA in a rod-shaped capsid. The viral life cycle includes three major phases: (1) early (or virus synthesis) phase (0.5-6 h after infection), which includes viral attachment, uncoating early viral gene expression, and shutting off host gene expression; (2) late phase (6-12 h after infection), which includes viral replication and formation of budded virus containing host plasma membrane and glycoprotein (gp)64, which is required for virus entry; and (3) very late phase (24-96 h after infection), which includes formation of polyhedral inclusion bodies and cell lysis...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510128/isolation-of-the-rna-cross-linking-immunoprecipitation-clip-tags-5-linker-ligation-reverse-transcription-polymerase-chain-reaction-rt-pcr-amplification-and-sequencing
#5
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This protocol describes purification of RNA cross-linking immunoprecipitation (CLIP) tags by proteinase K digestion of the cross-linked protein, addition of a 5' linker to the RNA tags, and amplification of the product by transcription-polymerase chain reaction (RT-PCR). Use of this protocol adds another important purification step: sizing of the PCR products to enrich for those derived from RNA originally cross-linked to the desired RNABP. Finally, sequencing of the PCR products is described. There are two strategies for sequencing the PCR products of "CLIPed" RNA...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510127/3-linker-ligation-and-size-selection-by-sds-page-for-cross-linking-immunoprecipitation-clip
#6
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3' linker loses the puromycin blocking group, concatemerization of the 3' linker will occur during the 3' linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Additionally, this protocol describes the treatment of coimmunoprecipitated RNA tags for CLIP with alkaline phosphatase to remove the 3' phosphate remaining after RNase digestion...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510126/immunoprecipitation-and-sds-page-for-cross-linking-immunoprecipitation-clip
#7
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This first part of this protocol is designed to optimize purification of the RNABP by immunoprecipitation for cross-linking immunoprecipitation (CLIP) experiments. The key variables to assess are the quality and quantity of antibody needed to immunoprecipitate most but not quite all of the RNABP (the titration will decrease nonspecific binding), and the tolerance of the antibody:antigen interaction to stringent wash conditions. The results of these experiments can be checked first by western blot, and subsequently using the pilot CLIP protocol described in the second half of this protocol...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510125/ultraviolet-uv-cross-linking-of-live-cells-lysate-preparation-and-rnase-titration-for-cross-linking-immunoprecipitation-clip
#8
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
One of the great advantages of RNA CLIP (cross-linking immunoprecipitation) is that RNA-protein complexes can be "frozen" in situ in live cells by ultraviolet (UV) irradiation. This protocol describes UV cross-linking of mammalian tissue culture cells or whole tissues. For the latter, the tissue is typically triturated to allow UV penetration. However, depending on the thickness of the chosen tissue, this may not be necessary. It is preferable to handle the tissue as little as possible, to keep it in ice-cold buffers, and to cross-link as soon after the time of collection as is feasible to preserve native interactions at the time of cross-linking...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30510124/in-vitro-screen-to-identify-silent-but-activatable-s-a-integration-sites-for-a-tetracycline-inducible-transgene-in-mice
#9
Richard Behringer, Marina Gertsenstein, Kristina Vintersten Nagy, Andras Nagy
To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the tet-O promoter. Therefore, the challenge of getting this system working properly is a serious prospect...
December 3, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385676/fluorescent-cell-staining-methods-for-living-hypsibius-exemplaris-embryos
#10
Kristen M McGreevy, Kira L Heikes, Shiri Kult, Marla E Tharp, Bob Goldstein
The tardigrade Hypsibius exemplaris was chosen as a model system in part because animals and embryos are optically clear at all stages, facilitating the visualization of events in living material. Here we report new methods for introducing fluorescent dyes into developing H. exemplaris embryos, including methods for fluorescently marking mitochondria, lysosomes, membranes, and nuclei. The development of these techniques suggests approaches for attempting to introduce other molecules into embryos.
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385675/total-rna-extraction-from-tardigrades
#11
Thomas C Boothby
Purification of high-quality total RNA from specimens is essential for many molecular techniques. In tardigrades (water bears), disruption of the cuticle is an important step in obtaining a good yield that is representative of all tissues of the animal. As with all single-stranded RNA methods, sterile technique, proper storage conditions, and handling are required for maintaining the quality and integrity of the material. This procedure will result in high-quality total RNA suitable for downstream applications such as cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), RNAseq library generation, and northern blots...
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385674/microinjection-of-dsrna-in-tardigrades
#12
Jennifer R Tenlen
Classical genetic analysis in the tardigrade Hypsibius exemplaris is a challenge because these animals are parthenogens. The publication of the H. exemplaris genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in Caenorhabditis elegans. A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5' end of each strand...
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385673/embryonic-in-situ-hybridization-for-the-tardigrade-hypsibius-exemplaris
#13
Frank W Smith
In situ hybridization is a method for visualizing embryonic gene expression that is amenable to nonmodel systems. Here, an in situ hybridization protocol is presented for the tardigrade Hypsibius exemplaris This method allows gene expression to be visualized directly and with fluorescence microscopy.
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385672/embryonic-immunostaining-for-the-tardigrade-hypsibius-exemplaris
#14
Frank W Smith, Willow N Gabriel
Immunostaining is a method used to visualize the localization of proteins in fixed tissue. Many antibodies are available that recognize specific proteins in a wide diversity of organisms, which makes this method ideal for investigating gene expression patterns in nonmodel animal systems. This protocol describes immunostaining for studies of embryogenesis in the tardigrade Hypsibius exemplaris .
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385671/live-imaging-of-tardigrade-embryonic-development-by-differential-interference-contrast-microscopy
#15
Kira L Heikes, Bob Goldstein
The tardigrade Hypsibius exemplaris was chosen as a model system in part because embryos and animals are optically clear at all stages, facilitating the viewing and filming of internal processes. Multiplane video recordings under differential interference contrast (DIC) microscopy have allowed early embryonic cell lineages to be reconstructed through seven rounds of division and have revealed invariant patterns of asymmetric cell divisions, nuclear migrations, and cell migrations. Here, we present a protocol for filming embryonic development of H...
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385670/desiccation-of-hypsibius-exemplaris
#16
Thomas C Boothby
Many species of tardigrades can survive severe water loss, but different species tolerate different desiccation conditions. Hypsibius exemplaris is able to survive desiccation after an initial period of slow drying, as described here. This protocol will likely work for other tardigrade species as well. Drying of tardigrades can be used for probing the mechanistic underpinnings of desiccation tolerance, as well as for practical purposes such as shipping and long-term storage of the animals.
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385669/laboratory-culture-of-hypsibius-exemplaris
#17
Robert McNuff
I have reared a culture of the tardigrade Hypsibius exemplaris for 30 years, since 1987. Here, I present my culture protocol.
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30385668/the-emergence-of-the-tardigrade-hypsibius-exemplaris-as-a-model-system
#18
Bob Goldstein
The success of scientists in revealing biological mechanisms has depended in large part on choosing tractable model systems. In 1997, molecular phylogenetics revealed that two of biology's most tractable models- Caenorhabditis elegans and Drosophila -are much more closely related to each other than had been thought previously. I began to explore whether any of the little-studied members of this branch of the tree of life might serve as a new model for comparative biology that could make use of the rich and ongoing sources of information flowing from C...
November 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30275081/analysis-and-normalization-of-real-time-polymerase-chain-reaction-pcr-experimental-data
#19
Michael R Green, Joseph Sambrook
In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template...
October 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30275080/making-weak-antigens-strong-cross-linking-peptides-to-klh-with-maleimide
#20
Edward A Greenfield, James DeCaprio, Mohan Brahmandam
Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. KLH-hapten conjugates are commonly used in antibody generation in a variety of hosts such as mice, rats, and rabbits...
October 1, 2018: Cold Spring Harbor Protocols
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