Fluorescence Microscopy-Based RNA Interference Screening

2014 ◽  
pp. 59-66 ◽  
Author(s):  
Manuel Gunkel ◽  
Nina Beil ◽  
Jürgen Beneke ◽  
Jürgen Reymann ◽  
Holger Erfle
Keyword(s):  
Rna Interference ◽  
Fluorescence Microscopy

The Research of Specific Inhibition of Survivin Expression To Reverse Drug Resistance with RNA Interference Technology.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5065-5065
Author(s):  
Juan Li ◽  
Ying Zhao ◽  
Shao-kai Luo ◽  
Dian-bao Zhang ◽  
Bei-hui Haung
Keyword(s):  
Drug Resistance ◽  
Rna Interference ◽  
Fluorescence Microscopy ◽  
Protein Expression ◽  
Cell Line ◽  
Survivin Expression ◽  
Survivin Mrna ◽  
Mrna Transcription ◽  
Before And After

Abstract Objective:Use RNA interference technology to down-regulate the expression of survivin gene in KM3 cells, observe its induction of apoptosis of KM3 cells, and if it can increase chemotherapy sensitivity of KM3 cells to adriamycin. Methods: SiRNA designed and in-vitro chemosynthesized aiming to survivin was transfected into human myeloma cell line KM3 mediated by liposome. Survivin mRNA transcription and protein expression of KM3 cells were then detected using RT-PCR and Western-blot 24, 48 and 72 hours after transfection, so as to invest the silencing effect. Besides, the apoptosis of the cell line were observed under fluorescence microscopy before and after transfection. Furthermore, cytotoxic analysis was used to compare the sensitivity variation of KM3 cells to adriamycin before and after transfection. Results: Down-regulations of survivin mRNA transcription and protein expression of KM3 cells could be observed 24 hours after siRNA tranfection, which were (12±2.3)% and (9.4±1.8)% respectively. After 48 hours, they were (61.4±7.9)% and (38.6±6.7)% respectively, while the survivin mRNA transcription and protein expression was increased quickly 72 hours later. Under fluorescence microscopy, the apoptosis rate of KM3 cells transfected with survivin siRNA 48 hours later was 28±7%, which was significantly higher than the control cells. (P<0.05). The IC50 value of KM3 cells to adriamycin decreased from 1.12±0.14uM to 0.21±0.03uM, which means the sensitivity of KM3 cells to adriamycin was 5.3 fold increased. Conclusions: SiRNA which was designed and synthesized to target survivin could effectively down-regulate survivin mRNA transcription and protein expression in KM3 cells, induce KM3 cells to apoptosis, increase its sensitivity to adriamycin after down-regulation, and effectively reverse drug-resistance of MM cells.

Cellular Phenotype Recognition for High-Content RNA Interference Genome-Wide Screening

2007 ◽  
Vol 13 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Jun Wang ◽  
Xiaobo Zhou ◽  
Pamela L. Bradley ◽  
Shih-Fu Chang ◽  
Norbert Perrimon ◽  
...  
Keyword(s):  
Rna Interference ◽  
Fluorescence Microscopy ◽  
Cultured Cells ◽  
Function Analysis ◽  
Small Gtpases ◽  
Biological Information ◽  
High Content Screening ◽  
Cellular Phenotype ◽  
Genome Wide ◽  
Cellular Phenotypes

Genome-wide, cell-based screens using high-content screening (HCS) techniques and automated fluorescence microscopy generate thousands of high-content images that contain an enormous wealth of cell biological information. Such screens are key to the analysis of basic cell biological principles, such as control of cell cycle and cell morphology. However, these screens will ultimately only shed light on human disease mechanisms and potential cures if the analysis can keep up with the generation of data. A fundamental step toward automated analysis of high-content screening is to construct a robust platform for automatic cellular phenotype identification. The authors present a framework, consisting of microscopic image segmentation and analysis components, for automatic recognition of cellular phenotypes in the context of the Rho family of small GTPases. To implicate genes involved in Rac signaling, RNA interference (RNAi) was used to perturb gene functions, and the corresponding cellular phenotypes were analyzed for changes. The data used in the experiments are high-content, 3-channel, fluorescence microscopy images of Drosophila Kc167 cultured cells stained with markers that allow visualization of DNA, polymerized actin filaments, and the constitutively activated Rho protein RacV12. The performance of this approach was tested using a cellular database that contained more than 1000 samples of 3 predefined cellular phenotypes, and the generalization error was estimated using a cross-validation technique. Moreover, the authors applied this approach to analyze the whole high-content fluorescence images of Drosophila cells for further HCS-based gene function analysis. ( Journal of Biomolecular Screening 2008:29-39)

scholarly journals Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

2013 ◽  
Vol 12 (7) ◽  
pp. 970-978 ◽  
Author(s):  
Kirsten Beck ◽  
Nathalie Acestor ◽  
Anjelique Schulfer ◽  
Atashi Anupama ◽  
Jason Carnes ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Rna Interference ◽  
Fluorescence Microscopy ◽  
Trypanosoma Brucei ◽  
Cell Population ◽  
Growth Defect ◽  
Kinetoplast Dna ◽  
Multiprotein Complexes ◽  
Proteomic Approach ◽  
Novel Protein

ABSTRACT The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

scholarly journals A laboratory module that explores RNA interference and codon optimization through fluorescence microscopy using Caenorhabditis elegans

2020 ◽  
Author(s):  
Maryam A. Azmi ◽  
Nicholas J. Palmisano ◽  
Taylor N. Medwig-Kinney ◽  
Frances E. Moore ◽  
Rumana Rahman ◽  
...  
Keyword(s):  
Image Analysis ◽  
Critical Thinking ◽  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Fluorescence Microscopy ◽  
Codon Optimization ◽  
List Type ◽  
Fluorescent Intensity ◽  
C Elegans ◽  
Publication Quality

AbstractAuthentic research experiences are beneficial to students allowing them to gain laboratory and problem-solving skills as well as foundational research skills in a team-based setting. We designed a laboratory module to provide an authentic research experience to stimulate curiosity, introduce students to experimental techniques, and promote higher-order thinking. In this laboratory module, students learn about RNA interference (RNAi) and codon optimization using the research organism Caenorhabditis elegans (C. elegans). Students are given the opportunity to perform a commonly used method of gene downregulation in C. elegans where they visualize gene depletion using fluorescence microscopy and quantify the efficacy of depletion using quantitative image analysis. The module presented here educates students on how to report their results and findings by generating publication quality figures and figure legends. The activities outlined exemplify ways by which students can acquire the critical thinking, data interpretation, and technical skills, which are beneficial for future laboratory classes, independent inquiry-based research projects and careers in the life sciences and beyond.SCIENTIFIC TEACHING CONTENTLearning GoalsGain experience working with C. elegansUnderstand the process of RNA interference and importance of codon optimizationLearn basic microscopy techniques and image analysisLearn how to properly use the scientific methodEnhance critical thinking skillsLearning ObjectivesStudents will be able to:Lab 1 and 2: Identify specific larval stages of C. elegansSynchronize C. elegans larvae using alkaline hypochlorite treatmentUnderstand codon usageFormulate hypotheses and design a controlled experimentLab 3 and 4: Acquire images using an epifluorescence microscopeEffectively communicate results and formulate conclusions from dataDescribe what RNAi is and how it affects gene expression/activityCalculate mean fluorescent intensity from acquired fluorescence micrographsPerform statistical tests to determine the significance of resultsGenerate publication quality figures and figure legends

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Localization of Immune Complexes in the Basement Membrane of the Ductuli Efferentes of the Rhesus Monkey

1980 ◽  
Vol 38 ◽  
pp. 456-457
Author(s):  
D. Marsh
Keyword(s):  
Basement Membrane ◽  
Fluorescence Microscopy ◽  
Rhesus Monkey ◽  
Immune Complex ◽  
Immune Complexes ◽  
Vas Deferens ◽  
Frozen Sections ◽  
Efferent Ducts ◽  
Complex Deposition ◽  
Sperm Antibodies

As a result of vasectomy, spermatozoa are confined to the epididymis and vas deferens, where they degenerate, releasing antigens that enter the circulation or are engulfed by macrophages. Multiple antigens of the sperm can elicit production of autoantibodies; circulating anti-sperm antibodies are found in a large percentage of vasectomized men, indicating the immunogenicity of the sperm. The increased prevalence of macrophages in the liomen of the rhesus monkey testicular efferent ducts after vasectomy led to further study of this region. Frozen sections were used for evaluation of immunopathological status by fluorescence microscopy with fluorescein-conjugated antibody. Subsequent granular deposits of immune complexes were revealed by positive immunofluorescence staining for complement. The immune complex deposition in the basement membrane surrounding the efferent ducts implies that this region is involved in antigen leakage (Fig. 1).

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