Molecular Biology Techniques Quiz
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Questions and Answers

The fluorescent signal is specific to the target dsDNA.

False (B)

The TaqMan method utilizes sequence-specific probes that can emit a fluorescent signal.

True (A)

SYBER green is specific only to the target dsDNA.

False (B)

When the probe in the TaqMan method is intact, it emits fluorescence.

<p>False (B)</p> Signup and view all the answers

The fluorescent signal released in the TaqMan method correlates with the quantity of target copies present.

<p>True (A)</p> Signup and view all the answers

Gene Expression Micro Array analyzes DNA directly without reverse transcription.

<p>False (B)</p> Signup and view all the answers

The TaqMan method is less specific than other detection methods.

<p>False (B)</p> Signup and view all the answers

Multiple samples can be analyzed simultaneously using multi-well systems.

<p>True (A)</p> Signup and view all the answers

Multiplexing allows for the detection of multiple protein markers in a single sample using different secondary antibodies.

<p>True (A)</p> Signup and view all the answers

The only method to detect protein markers in a sample is through antibody-based approaches.

<p>False (B)</p> Signup and view all the answers

Flow cytometry characterizes cells individually as they pass through a laser beam in a fluid phase.

<p>True (A)</p> Signup and view all the answers

High content imaging can only analyze a single well at a time during experiments.

<p>False (B)</p> Signup and view all the answers

Immunostaining is primarily a quantitative technique for assessing protein presence in samples.

<p>False (B)</p> Signup and view all the answers

Fluorescence-based detection methods can be amplified through enzymatic-based reactions.

<p>False (B)</p> Signup and view all the answers

False positives can be avoided by using appropriate controls in protein detection experiments.

<p>True (A)</p> Signup and view all the answers

Simultaneous immunodetection requires the use of incompatible labels.

<p>False (B)</p> Signup and view all the answers

The barcode used in the DSP technology can consist of three colors.

<p>False (B)</p> Signup and view all the answers

The procedure involves aspirating and collecting the barcodes in the region of interest (ROI) for analysis.

<p>True (A)</p> Signup and view all the answers

Multiplex analysis using Ab-barcode and RNA complementary sequence-barcode can provide limited information.

<p>False (B)</p> Signup and view all the answers

The recognition site of the tag in 2nd generation technology can cover more than one gene.

<p>True (A)</p> Signup and view all the answers

Fluorescent reporters are used to bind to the recognition domain and provide visual feedback.

<p>True (A)</p> Signup and view all the answers

UV light is used to enhance the binding of the barcodes to their respective probes.

<p>False (B)</p> Signup and view all the answers

In the first generation of the technology, separating the target complimentary sequence from the DSP barcode is necessary.

<p>True (A)</p> Signup and view all the answers

The digital spatial profiler (DSP) technology cannot analyze targets directly on the sample.

<p>False (B)</p> Signup and view all the answers

CDNA samples are hybridized onto a probe array that contains thousands of sequences corresponding to different genes.

<p>True (A)</p> Signup and view all the answers

The washing procedures after hybridization enhance the attachment of cDNA to the probes.

<p>False (B)</p> Signup and view all the answers

Red colored signals on a microarray slide indicate that both mRNA samples express the same gene.

<p>False (B)</p> Signup and view all the answers

Nanostring technology involves immobilizing the probe onto a slide before hybridizing with the target gene in a liquid phase.

<p>False (B)</p> Signup and view all the answers

By analyzing the colors emitted by the cDNA tags, researchers can determine which genes are represented on the microarray slide.

<p>True (A)</p> Signup and view all the answers

The detection of cDNA involves scanning the slide to identify positions that emit fluorescent signals.

<p>True (A)</p> Signup and view all the answers

The introduction of different fluorescent-labeled nucleotides allows comparison of gene expression levels across various mRNA samples.

<p>True (A)</p> Signup and view all the answers

Microarray technology can be used in clinical settings to analyze the expression of RNA in benign tissues only.

<p>False (B)</p> Signup and view all the answers

The technique called FISH is applied exclusively to RNA samples.

<p>False (B)</p> Signup and view all the answers

A negative control in hybridization experiments is a probe complementary to the target mRNA.

<p>False (B)</p> Signup and view all the answers

Multiplexed target surveys can utilize the RNAscope method to achieve single RNA molecule marking.

<p>True (A)</p> Signup and view all the answers

Fluorochromes are one type of tag that can be attached to dNTPs for RNA probe synthesis.

<p>True (A)</p> Signup and view all the answers

ISH techniques are ineffective for localization of RNA transcripts because they require high abundances.

<p>True (A)</p> Signup and view all the answers

The process of probe hybridization occurs only if the tissue/sample contains the target sequence.

<p>True (A)</p> Signup and view all the answers

The limitations of ISH include its application being efficient for a large number of genes simultaneously.

<p>False (B)</p> Signup and view all the answers

Immunostaining is one method used for probe detection in various hybridization techniques.

<p>True (A)</p> Signup and view all the answers

Integrins are able to bind only to intracellular proteins.

<p>False (B)</p> Signup and view all the answers

Actin filaments in the cytoplasm connect to integrins on the cell's surface.

<p>True (A)</p> Signup and view all the answers

Talin and vinculin are intermediary proteins that connect integrins and the actin cytoskeleton.

<p>True (A)</p> Signup and view all the answers

Integrins primarily recognize and bind to lipids found in the extracellular environment.

<p>False (B)</p> Signup and view all the answers

Integrins are transmembrane proteins involved in cell adhesion.

<p>True (A)</p> Signup and view all the answers

Cell behavior is unaffected by the nature of the surface the cell is adhering to.

<p>False (B)</p> Signup and view all the answers

The binding of integrins to ECM proteins is permanent and cannot be detached.

<p>False (B)</p> Signup and view all the answers

Intercellular connections can include E-cadherin and gap junctions.

<p>True (A)</p> Signup and view all the answers

Flashcards

Multiplexing

A technique that uses multiple antibodies labeled with different fluorescent tags to detect and identify different proteins within the same sample.

Antibody-based Detection

A method of protein detection that uses antibodies to specifically bind to and visualize a target protein in a sample.

Flow Cytometry

A technique that uses a light beam to illuminate individual cells in a fluid stream, allowing for the measurement and analysis of different cell properties.

Western Blot

A technique that separates proteins based on their size and charge, revealing the presence of specific proteins in a sample.

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Immunostaining

A technique that uses antibodies to detect the presence of a specific protein within a sample, indicating whether the protein is present or absent.

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Multispectral Imaging

A type of imaging that uses multiple wavelengths of light to capture images of different fluorescent signals, allowing for the simultaneous analysis of multiple proteins.

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False Positive

The presence of a signal in a protein detection experiment that is not actually due to the target protein.

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False Negative

The absence of a signal in a protein detection experiment despite the target protein being present.

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TaqMan Method

A technique for measuring gene expression, where probes with specific sequences bind to target DNA and release a fluorescent signal when cleaved by DNA polymerase.

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SYBR Green

A method for detecting the amount of DNA in a sample. It uses the fluorescent dye SYBR Green, which binds to double-stranded DNA, increasing its signal intensity.

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Fluorescence Quenching

The use of a probe with a quencher that prevents fluorescence until cleaved by polymerase during DNA replication.

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Reverse Transcription

The process of converting RNA into cDNA to be used for further analysis. Often used in gene expression studies as it allows measuring mRNA levels.

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Multi-well Systems

A technique that allows simultaneous analysis of multiple samples using a multi-well plate.

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Gene Expression Microarrays

A method for studying gene expression by analyzing the mRNA levels of a large number of genes simultaneously.

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Marker Survey

A technique used to survey the expression of a group of genes in a sample, usually using a gene expression microarray.

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Marker

The type of gene that is measured in a marker survey, providing information about the biological activity of the sample.

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Integrins

Special receptor proteins that bind to extracellular matrix (ECM) proteins, acting as sensors and anchors for cells.

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Actin

The protein that anchors integrins inside the cell, connecting them to the actin cytoskeleton.

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Intermediary proteins (talin, vinculin)

Proteins that bridge the gap between integrins and the actin cytoskeleton, helping to strengthen the link.

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E-cadherin binding

The type of cell-to-cell connection where cells bind tightly together through E-cadherin proteins.

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Occludin/Claudin binding

The type of cell-to-cell connection where cells bind tightly together through Occludin/Claudin proteins, creating a tight seal that prevents leakage.

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Gap junctions

The type of cell-to-cell connection that allows direct communication between cells through channels.

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Seeding Surfaces

The process by which cells respond to the surface they're attached to.

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Integrin signaling

The ability of integrins to bind to ECM proteins and signal the cell to adapt to its environment.

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In Situ Hybridization (ISH)

A technique used to detect and visualize specific nucleic acid sequences (DNA or RNA) within cells or tissues.

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RNA In Situ Hybridization (RISH)

In Situ Hybridization using RNA probes to detect and visualize specific RNA sequences within cells or tissues.

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DNA In Situ Hybridization (DISH)

In Situ Hybridization using DNA probes to detect and visualize specific DNA sequences within cells or tissues.

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Fluorescence In Situ Hybridization (FISH)

A type of DISH that employs fluorescently labeled probes for visualization.

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Probe

A short sequence of nucleic acid (DNA or RNA) that is designed to bind to a complementary target sequence.

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RNAscope

A method that uses a series of paired probes and signal amplification to mark single RNA molecules in a tissue or cell culture.

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Digital Spatial Profiling (DSP)

A method for analyzing the spatial distribution of RNA and proteins in a tissue sample.

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Region of Interest (ROI)

A region of interest (ROI) is a specific area within a tissue sample that is selected for analysis.

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Negative Control in ISH

A negative control in ISH experiments to ensure that the observed signal is specific to the target sequence.

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DNA FISH Applications

A technique used to detect and visualize specific sequences in the genome, perform karyotypic diagnostics, and track cells in vivo.

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DSP (Digital Spatial Profiler)

A digital spatial profiler (DSP) is a barcode attached to a probe used in DSP technology. It consists of a unique sequence of two colors for identification.

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Probe Cleavage

In DSP, the process of breaking down the probe by UV light to separate the target complementary sequence and the DSP barcode.

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Barcode Analysis

The collection and analysis of all DSP barcodes released from the ROI after probe cleavage, which allows for a list of detected molecules.

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2nd Generation DSP

A technique that allows the analysis of DSP barcodes directly on the tissue sample without detaching them. This is done by using tags with a recognition domain and a readout domain.

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Recognition Domain

The recognition site of a tag in 2nd generation DSP can recognize a large part of a target molecule, covering multiple genes or protein regions.

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Microarray Analysis

A technique that uses a microarray slide to detect the presence and amount of specific mRNA transcripts. Thousands of different probes, each representing a unique gene, are attached to the slide. The sample's cDNA, labeled with fluorescent tags, is hybridized to the array. Only cDNAs complementary to their corresponding probes bind, and the intensity of the fluorescence indicates the level of expression of the gene.

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Why cDNA?

Complementary DNA (cDNA) is generated from mRNA through reverse transcription. This process allows the analysis of gene expression as mRNA is unstable, while cDNA is more stable.

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Fluorescent Tags in Microarrays

Fluorescent tags, such as dyes or probes, are attached to cDNA strands. The intensity of the fluorescence signal indicates the abundance of the corresponding mRNA.

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Comparing Expression Levels

By comparing fluorescence intensities across different samples, researchers can identify differences in gene expression levels.

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Two-Color Microarrays

In a two-color microarray, each sample's cDNA is labeled with a different fluorescent dye, allowing for the simultaneous comparison of gene expression between two samples. The color combinations reveal whether genes are upregulated, downregulated, or unchanged in each sample.

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Nanostring Technology

The Nanostring Technology uses probes that bind to target genes in a liquid phase before being immobilized on a slide. This allows for the identification and quantification of specific mRNA transcripts.

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Single Experiment Analysis

Nanostring analyzes RNA transcripts in a single experiment, providing information about the abundance of specific RNA transcripts in the sample.

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Applications of Nanostring

Nanostring is utilized in various applications, including gene expression profiling, biomarker discovery, and cancer research.

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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

  • Direct morphogen gradients involve a localized signal source and diffusing molecules forming a gradient

  • Indirect gradients exploit inhibitor signals diffusing away from a source, influencing signal gradient activity.

  • Receptor-mediated signaling involves an extracellular molecule interacting with a receptor protein, triggering intracellular signaling cascades

  • 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.

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