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Questions and Answers
The fluorescent signal is specific to the target dsDNA.
The fluorescent signal is specific to the target dsDNA.
False
The TaqMan method utilizes sequence-specific probes that can emit a fluorescent signal.
The TaqMan method utilizes sequence-specific probes that can emit a fluorescent signal.
True
SYBER green is specific only to the target dsDNA.
SYBER green is specific only to the target dsDNA.
False
When the probe in the TaqMan method is intact, it emits fluorescence.
When the probe in the TaqMan method is intact, it emits fluorescence.
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The fluorescent signal released in the TaqMan method correlates with the quantity of target copies present.
The fluorescent signal released in the TaqMan method correlates with the quantity of target copies present.
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Gene Expression Micro Array analyzes DNA directly without reverse transcription.
Gene Expression Micro Array analyzes DNA directly without reverse transcription.
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The TaqMan method is less specific than other detection methods.
The TaqMan method is less specific than other detection methods.
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Multiple samples can be analyzed simultaneously using multi-well systems.
Multiple samples can be analyzed simultaneously using multi-well systems.
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Multiplexing allows for the detection of multiple protein markers in a single sample using different secondary antibodies.
Multiplexing allows for the detection of multiple protein markers in a single sample using different secondary antibodies.
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The only method to detect protein markers in a sample is through antibody-based approaches.
The only method to detect protein markers in a sample is through antibody-based approaches.
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Flow cytometry characterizes cells individually as they pass through a laser beam in a fluid phase.
Flow cytometry characterizes cells individually as they pass through a laser beam in a fluid phase.
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High content imaging can only analyze a single well at a time during experiments.
High content imaging can only analyze a single well at a time during experiments.
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Immunostaining is primarily a quantitative technique for assessing protein presence in samples.
Immunostaining is primarily a quantitative technique for assessing protein presence in samples.
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Fluorescence-based detection methods can be amplified through enzymatic-based reactions.
Fluorescence-based detection methods can be amplified through enzymatic-based reactions.
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False positives can be avoided by using appropriate controls in protein detection experiments.
False positives can be avoided by using appropriate controls in protein detection experiments.
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Simultaneous immunodetection requires the use of incompatible labels.
Simultaneous immunodetection requires the use of incompatible labels.
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The barcode used in the DSP technology can consist of three colors.
The barcode used in the DSP technology can consist of three colors.
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The procedure involves aspirating and collecting the barcodes in the region of interest (ROI) for analysis.
The procedure involves aspirating and collecting the barcodes in the region of interest (ROI) for analysis.
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Multiplex analysis using Ab-barcode and RNA complementary sequence-barcode can provide limited information.
Multiplex analysis using Ab-barcode and RNA complementary sequence-barcode can provide limited information.
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The recognition site of the tag in 2nd generation technology can cover more than one gene.
The recognition site of the tag in 2nd generation technology can cover more than one gene.
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Fluorescent reporters are used to bind to the recognition domain and provide visual feedback.
Fluorescent reporters are used to bind to the recognition domain and provide visual feedback.
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UV light is used to enhance the binding of the barcodes to their respective probes.
UV light is used to enhance the binding of the barcodes to their respective probes.
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In the first generation of the technology, separating the target complimentary sequence from the DSP barcode is necessary.
In the first generation of the technology, separating the target complimentary sequence from the DSP barcode is necessary.
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The digital spatial profiler (DSP) technology cannot analyze targets directly on the sample.
The digital spatial profiler (DSP) technology cannot analyze targets directly on the sample.
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CDNA samples are hybridized onto a probe array that contains thousands of sequences corresponding to different genes.
CDNA samples are hybridized onto a probe array that contains thousands of sequences corresponding to different genes.
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The washing procedures after hybridization enhance the attachment of cDNA to the probes.
The washing procedures after hybridization enhance the attachment of cDNA to the probes.
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Red colored signals on a microarray slide indicate that both mRNA samples express the same gene.
Red colored signals on a microarray slide indicate that both mRNA samples express the same gene.
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Nanostring technology involves immobilizing the probe onto a slide before hybridizing with the target gene in a liquid phase.
Nanostring technology involves immobilizing the probe onto a slide before hybridizing with the target gene in a liquid phase.
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By analyzing the colors emitted by the cDNA tags, researchers can determine which genes are represented on the microarray slide.
By analyzing the colors emitted by the cDNA tags, researchers can determine which genes are represented on the microarray slide.
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The detection of cDNA involves scanning the slide to identify positions that emit fluorescent signals.
The detection of cDNA involves scanning the slide to identify positions that emit fluorescent signals.
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The introduction of different fluorescent-labeled nucleotides allows comparison of gene expression levels across various mRNA samples.
The introduction of different fluorescent-labeled nucleotides allows comparison of gene expression levels across various mRNA samples.
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Microarray technology can be used in clinical settings to analyze the expression of RNA in benign tissues only.
Microarray technology can be used in clinical settings to analyze the expression of RNA in benign tissues only.
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The technique called FISH is applied exclusively to RNA samples.
The technique called FISH is applied exclusively to RNA samples.
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A negative control in hybridization experiments is a probe complementary to the target mRNA.
A negative control in hybridization experiments is a probe complementary to the target mRNA.
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Multiplexed target surveys can utilize the RNAscope method to achieve single RNA molecule marking.
Multiplexed target surveys can utilize the RNAscope method to achieve single RNA molecule marking.
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Fluorochromes are one type of tag that can be attached to dNTPs for RNA probe synthesis.
Fluorochromes are one type of tag that can be attached to dNTPs for RNA probe synthesis.
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ISH techniques are ineffective for localization of RNA transcripts because they require high abundances.
ISH techniques are ineffective for localization of RNA transcripts because they require high abundances.
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The process of probe hybridization occurs only if the tissue/sample contains the target sequence.
The process of probe hybridization occurs only if the tissue/sample contains the target sequence.
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The limitations of ISH include its application being efficient for a large number of genes simultaneously.
The limitations of ISH include its application being efficient for a large number of genes simultaneously.
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Immunostaining is one method used for probe detection in various hybridization techniques.
Immunostaining is one method used for probe detection in various hybridization techniques.
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Integrins are able to bind only to intracellular proteins.
Integrins are able to bind only to intracellular proteins.
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Actin filaments in the cytoplasm connect to integrins on the cell's surface.
Actin filaments in the cytoplasm connect to integrins on the cell's surface.
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Talin and vinculin are intermediary proteins that connect integrins and the actin cytoskeleton.
Talin and vinculin are intermediary proteins that connect integrins and the actin cytoskeleton.
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Integrins primarily recognize and bind to lipids found in the extracellular environment.
Integrins primarily recognize and bind to lipids found in the extracellular environment.
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Integrins are transmembrane proteins involved in cell adhesion.
Integrins are transmembrane proteins involved in cell adhesion.
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Cell behavior is unaffected by the nature of the surface the cell is adhering to.
Cell behavior is unaffected by the nature of the surface the cell is adhering to.
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The binding of integrins to ECM proteins is permanent and cannot be detached.
The binding of integrins to ECM proteins is permanent and cannot be detached.
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Intercellular connections can include E-cadherin and gap junctions.
Intercellular connections can include E-cadherin and gap junctions.
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Study Notes
Cell Signaling Principles
- Cell phenotype is determined by the expression of genes, leading to mRNA and protein production. Different cell types have unique gene and protein expression profiles.
- Development has two aspects: growth and patterning. Patterning is controlled via cell signaling between different cell types.
- Cell proliferation, specialization, interaction, and movement are crucial aspects of development.
- The zygote (fertilized egg) produces three germ layers (ectopderm, mesoderm, endoderm) forming the basic tissue types.
Generating Cell Diversity
- Cells acquire diverse fates through intrinsic information from precursor cells.
- As cells divide, segregation of internal components (mRNA, cytokines) leads to varying patterns across daughter cells, creating diversity.
- Cells have the same signaling potential. However, different concentrations of signaling molecules trigger diverse gene expression.
Models of Cell Diversity
- Diversity can arise from non-symmetrical separation of determinants within precursor cells.
- Diversity can arise from specific signals from surrounding cells or other cells (interactions).
- In the "flag model", cells have the potential to develop into different types, with differing concentration levels of signaling triggering different differentiation outcomes.
Types of Signaling
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Direct morphogen gradients involve a localized signal source and diffusing molecules forming a gradient
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Indirect gradients exploit inhibitor signals diffusing away from a source, influencing signal gradient activity.
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Receptor-mediated signaling involves an extracellular molecule interacting with a receptor protein, triggering intracellular signaling cascades
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Signaling cascades often result in changes in gene expression and cell function.
Signaling Pathways
- Wnt, TGF-β, and Hedgehog pathways are important in development and other biological processes.
- Wnt signaling regulates the body axis, midbrain and haematopoiesis.
- TGF-β signaling is important for body axis alignment & skeleton/organ formation.
- Hedgehog signaling controls the patterning of the developing body, the left-right asymmetry, and development of the neural tube.
Cell Cycle Analysis
- CDK and cyclin complexes regulate cell cycle progression.
- DNA replication occurs during the S phase of the cell cycle.
- Telomeres, which shorten with each division, limit a cell's replicative potential (crisis).
- Telomerase activation permits a cell's continued proliferation (as seen in some cancers).
- Physiological factors such as metabolic stimuli, confluency, and chemical compounds can stop proliferation.
- Cell growth/proliferation measurements include counting cells, population doubling time, live imaging, protein quantification, and DNA quantification.
Cell Cycle Analysis Techniques
- Live imaging allows viewing how much surface is covered by cells over time.
- Protein quantification can be used to estimate cell numbers DNA quantification can be used to estimate cell numbers
Cell Cycle Analysis
- Propidium iodide (PI) is a dye used to analyze cell cycle phases via flow cytometry, showing different amounts and proportion of DNA.
- Flow cytometry can quantify the proportion of cells in different phases of the cell cycle.
- Radioactive and non-radioactive nucleotide incorporation assays can be used to study DNA replication and the cell cycle stage.
Protein Marker Detection Techniques
- Protein markers can be identified using antibodies; these antibodies can identify target proteins present in a cell sample, their location and if it is expressing specific proteins.
- Proteomic techniques (protein extraction/ digestion (into peptides)/ analysis via Mass spectrometry) can identify a protein profile within a sample, to compare them and see their abundance levels.
- Antibody techniques can be used in western blotting and Immunostaining can detect specific target proteins in a sample in their location.
Immunodetection Approaches
- Cell surface, cytoplasmic/cytoskeletal, and nuclear markers can be used to recognize proteins.
- Bulk detection method/ western blotting or ELISA determines if the protein is present or not.
- Localized detection using immunostaining methods determines the localization of proteins.
- Single cell detection (flow cytometry or MACS) determines which cells express the protein.
- ELISA and Dot blot methods can identify specific target protein in a sample.
Multiplexing
- Multiplexing methods for detecting several different protein markers can employ different secondary antibodies and different fluorochromes for multiplexing, in a single assay.
Flow Cytometry
- Flow cytometry measures and analyzes individual cells in a fluid stream using lasers and detectors.
- Forward scatter (FSC) measures cell size, and side scatter (SSC) measures cell granularity.
- Fluorescent channels measure the fluorescent intensity of stained cells, yielding data on cell subpopulations and distributions across various cell cycle phases.
Flow Cytometry Analysis
- Histograms (single parameter) show the distribution of cell intensity of fluorescence as well as the number of cells exhibiting a certain fluorescence.
- Dot plots (multiple parameters) are useful to discriminate between subpopulations of cells/cell populations, based on multiple markers simultaneously.
Cell Sorting (FACS)
- Fluorescence-activated cell sorting (FACS) separates cells into different populations based on their surface markers and fluorescence properties.
- Cells are labeled by specific fluorochrome-conjugated antibodies to target specific molecules on the cell surface or inside.
- Different properties (such as marker expression level) can be captured and measured in real time allowing for their separation
- MACS is an alternative method that uses magnetic beads to sort cells.
Cell Migration
- Cell migration is regulated by cell phenotype and its environment
- Cell migration processes include adhesion, protrusion, and contraction via cytoskeletal reorganisation.
- Cell migration is important in various biological contexts, such as embryonic development, tissue repair and cancer metastasis.
Collective cell migration
- Migrating leader-follower: The leading cells guide the movement of the following cells.
- Contact inhibition of migration: Cells change their migration direction once they collide with other cells.
- Contact stimulation of migration: The cells with high density around migrate, others gets isolated and stop migrating.
How to Measure Cell Adhesion
- Static adhesion assays involve measuring how many cells adhere to a surface over time.
- Adhesion measurement uses force measurement to measure the cell-surface interaction strength.
- Washing techniques use stirrers to measure cell adhesion.
- Spinning disk assays use centrifugal force to separate adhered cells/non-adhered cells.
- Flow chamber assays use controlled flow to measure how cells adhere.
- Impedance measurement measures a change in the electronic flow/resistance as cells adhere.
Cell Migration Assays
- Scratch or wound-healing assays are in vitro models to measure how much a population of cells closes a scratch or gap on the tissue over time.
- 2D cell tracking assays track single cells over time to measure migration speed, direction, and other related parameters on a 2D substrate.
- microfluidic chambers mimic in vivo cell migration in a controlled experiment.
- Membrane migration assays measure how many cells migrate through a membrane.
- Transwell or boyden chambers measure cell invasion (ability to penetrate a membrane).
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Description
Test your knowledge on various molecular biology techniques, including the TaqMan method, SYBER Green, and flow cytometry. This quiz covers the principles, applications, and specifics of these methods in gene expression analysis and protein detection.
