scholarly journals A simple method for in situ, multiplexed measurement of RNA degradation by flow cytometry

2021 ◽  
Author(s):  
Jaya Rammohan ◽  
Nina Alperovich ◽  
Bin Shao ◽  
David Ross
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Biological Function ◽  
Rna Degradation ◽  
Rna Sequences ◽  
Simple Method ◽  
Rna Purification ◽  
Degradation Rates ◽  
Cellular Context

RNA degradation plays a major role in cellular function, but current methods for measuring RNA degradation require RNA purification or are low throughput. Here we show how a flow-FISH assay can be used for high-throughput, in situ measurement of RNA degradation without RNA purification. We demonstrate how this approach can be used to simultaneously measure RNA degradation rates of different RNA sequences in a single assay and explore how the assay can be used to examine the effect of cellular context on RNA degradation rates. This assay will be generally useful to quantitatively measure how natural and engineered biological function depends on RNA half-life.

Rotary Beveling for In Situ Thin-Section Microscopy of Cell Cultures

1981 ◽  
Vol 39 ◽  
pp. 550-551
Author(s):  
Dean A. Handley ◽  
Jack T. Alexander ◽  
Shu Chien
Keyword(s):  
Cell Growth ◽  
Cell Cultures ◽  
Molecular Interactions ◽  
Convenient Method ◽  
Petri Dish ◽  
Simple Method ◽  
Thin Section Microscopy ◽  
Thin Sectioning ◽  
Organic Fluids

In situ preparation of cell cultures for ultrastructural investigations is a convenient method by which fixation, dehydration and embedment are carried out in the culture petri dish. The in situ method offers the advantage of preserving the native orientation of cell-cell interactions, junctional regions and overlapping configurations. In order to section after embedment, the petri dish is usually separated from the polymerized resin by either differential cryo-contraction or solvation in organic fluids. The remaining resin block must be re-embedded before sectioning. Although removal of the petri dish may not disrupt the native cellular geometry, it does sacrifice what is now recognized as an important characteristic of cell growth: cell-substratum molecular interactions. To preserve the topographic cell-substratum relationship, we developed a simple method of tapered rotary beveling to reduce the petri dish thickness to a dimension suitable for direct thin sectioning.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

A Mild and Simple Method for Making MIDA Boronates

2020 ◽  
Author(s):  
Aidan Kelly ◽  
Peng-Jui (Ruby) Chen ◽  
Jenna Klubnick ◽  
Daniel J. Blair ◽  
Martin D. Burke
Keyword(s):  
Organic Synthesis ◽  
Boronic Acids ◽  
Simple Method ◽  
Direct Preparation ◽  
Synthesis Procedure ◽  
Harsh Conditions

<div> <div> <div> <p>Existing methods for making MIDA boronates require harsh conditions and complex procedures to achieve dehydration. Here we disclose that a pre-dried form of MIDA, MIDA anhydride, acts as both a source of the MIDA ligand and an in situ desiccant to enable a mild and simple MIDA boronate synthesis procedure. This method expands the range of sensitive boronic acids that can be converted into their MIDA boronate counterparts. Further utilizing unique properties of MIDA boronates, we have developed a MIDA Boronate Maker Kit which enables the direct preparation and purification of MIDA boronates from boronic acids using only heating and centrifuge equipment that is widely available in labs that do not specialize in organic synthesis. </p> </div> </div> </div>

scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

A deep learning-based concept for high throughput image flow cytometry

2021 ◽  
Vol 118 (12) ◽  
pp. 123701
Author(s):  
Julie Martin-Wortham ◽  
Steffen M. Recktenwald ◽  
Marcelle G. M. Lopes ◽  
Lars Kaestner ◽  
Christian Wagner ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
High Throughput ◽  
Image Flow

scholarly journals Comparison of Hydrocarbon-Degrading Consortia from Surface and Deep Waters of the Eastern Mediterranean Sea: Characterization and Degradation Potential

Energies ◽  
2021 ◽  
Vol 14 (8) ◽  
pp. 2246
Author(s):  
Georgia Charalampous ◽  
Efsevia Fragkou ◽  
Konstantinos A. Kormas ◽  
Alexandre B. De Menezes ◽  
Paraskevi N. Polymenakou ◽  
...  
Keyword(s):  
Mediterranean Sea ◽  
Oil Spills ◽  
Eastern Mediterranean ◽  
Sole Source ◽  
Eastern Mediterranean Sea ◽  
Deep Waters ◽  
Degradation Rates ◽  
Polycyclic Aromatic ◽  
Set Up

The diversity and degradation capacity of hydrocarbon-degrading consortia from surface and deep waters of the Eastern Mediterranean Sea were studied in time-series experiments. Microcosms were set up in ONR7a medium at in situ temperatures of 25 °C and 14 °C for the Surface and Deep consortia, respectively, and crude oil as the sole source of carbon. The Deep consortium was additionally investigated at 25 °C to allow the direct comparison of the degradation rates to the Surface consortium. In total, ~50% of the alkanes and ~15% of the polycyclic aromatic hydrocarbons were degraded in all treatments by Day 24. Approximately ~95% of the total biodegradation by the Deep consortium took place within 6 days regardless of temperature, whereas comparable levels of degradation were reached on Day 12 by the Surface consortium. Both consortia were dominated by well-known hydrocarbon-degrading taxa. Temperature played a significant role in shaping the Deep consortia communities with Pseudomonas and Pseudoalteromonas dominating at 25 °C and Alcanivorax at 14 °C. Overall, the Deep consortium showed a higher efficiency for hydrocarbon degradation within the first week following contamination, which is critical in the case of oil spills, and thus merits further investigation for its exploitation in bioremediation technologies tailored to the Eastern Mediterranean Sea.

scholarly journals On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 966
Author(s):  
Zuzana Chumová ◽  
Terezie Mandáková ◽  
Pavel Trávníček
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Crucial Role ◽  
Evolutionary History ◽  
Essential Role ◽  
Grass Species ◽  
Polyploid Species ◽  
Anthoxanthum Odoratum ◽  
European Populations

Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied.

Sign in / Sign up

Export Citation Format

Share Document