MIMM 414

Question 1

what is a viability dye

Answer 1

it stains cells that are damaged or dead
-aka only dead cells test positive

Question 2

what is the difference between full stains and single stains

Answer 2

single stains is when you stain for one antibody
-full stains is when you stain for all the antibodies

Question 3

what is an isotype control

Answer 3

-it controls for the specificity of the antibody and whether it has non specific binding to the fc receptor
-It requires that the isotype and the specific antibody have the same physiochemical properties and are labelled with the same molarity of fluorochrome.
-Typically the isotype control should have low binding, and should give only fluorescence intensities similar to that of unstained cells.

Question 4

what is an fmo control

Answer 4

fluorescence minus one; aka staining with all antibodies except one
-expected to show up as a negative control
-control is used to test of whether any fluorescence
spill over occurs. A comparison of a staining with an antibody mix containing the antibody (e.g. rabbit IgG1 anti-mouse IL-5 labelled with PE) vs without the antibodyis needed
-Typically there should be little fluorescent spillover if antibodies are used at correct concentrations and if
compensation is done properly

Question 5

what are compensation control

Answer 5

are used to adjust voltages in each channel of the flow cytometer prior to aquiring all other samples

Question 6

what is used to set gates that define cell populations of interest

Answer 6

fmo

Question 7

what is fsc

Answer 7

-forward scatter
-measure of cell size

Question 8

what is ssc

Answer 8

-side scatter
-measure of cell complexity aka granularity

Question 9

what is cytof

Answer 9

-mass cytometry technique similar to flow
-single cells are phenotyped via labelled antibodies
-antibodies are tagged with heavy metals instead of fluorophores and the metal tags are quantified by mass spec

Question 10

true or false: intracellular staining is only possible in cytof

Answer 10

false: it is also possible in flow cy

Question 11

what is an elisa

Answer 11

methods used for the quantification of molecules in solution by antibodies specific to the target molecule qith a colorimetric readout produced vy an enzyme conjugated to an antibody

Question 12

the heavy metal ions in cytof are quantified using what

Answer 12

time of flight mass spectrometry

Question 13

the quantity of reporter ions in a particular mass channel is a proxy for what

Answer 13

molecular expression

Question 14

what is the dimensionality reduction method used in cytof

Answer 14

visne

Question 15

differenced between flow and cytof

Answer 15

-mass is more precise because there is no compensation
-there is no need for single cell gating in mass
-there is no overlap in in mass
-mass breaks down the cells
-you can do over 40 cells in mass compared to flow

Question 16

what is atac-seq

Answer 16

assay for transposase accessible chromatin
-Assay for transposase-accessible chromatin sequencing (ATAC-seq) [9] is a technique that identifies open chromatin regions. A large Tn5 transposase complex carrying 2 adapter sequences is used to access regions of the genome that are “unraveled”.
-Tn5 will then cleave the double-stranded DNA and attach adapters such that fragments in open regions can be sequenced. After alignment of the sequencing reads to a reference genome, transcriptional activity in a cell or sample can be inferred from “peaks” of reads

Question 17

what is the role of chip seq

Answer 17

-identifies dna regions bound by a protein of interest
-they can be a TF or a histone with specific modification

Question 18

what is principal component analysis

Answer 18

-pca
-dimensionality reduction method
-it is defined by principal components

Question 19

what does pc1 and pc2 mean

Answer 19

it explains the most variance in the data

Question 20

draw backs from pca

Answer 20

-hard to interpret
-poor performance in very hihh dimensions
-highly sensitive to outliers
-unable yo capture nonlinear structure

Question 21

give some examples of dimensionality reduction methods

Answer 21

umap and s-sne

Question 22

what is tetramer staining

Answer 22

a technique in immunology that involves the use of fluorescently labeled tetramers—complexes of four major histocompatibility complex (MHC) molecules and specific peptide antigens. These tetramers can selectively bind to T cells that express corresponding T-cell receptors (TCRs) specific to the presented peptide.

Question 23

what is the spade analysis

Answer 23

represents cytometry data as a tree based on phenotypic similarities

Question 24

what does cre recombinase enzymes do

Answer 24

it recognizes a specific dna sequence called a loxP site
-giving access to 2 loxP sites the cre recombinase will excise the dna segments

Question 25

what is a floxed gene

Answer 25

it is flanked by 2 loxP sites

Question 26

true or false: you only need one generation to get cre lox mice

Answer 26

nah you need two to get homozygous things

Question 27

what does rna seq do

Answer 27

-RNA-seq (RNA sequencing) is a technique that aims to identify and quantify RNA molecules in a biological sample.
-basically it is to quantify gene expression levels

Question 28

what is scrna-seq

Answer 28

assessing gene expression on a single cell levelw

Question 29

what is tcr sequencing

Answer 29

-used to determine the clonalities of activated t cells in an immune response
-tcr epitope discovery methods identify antigens driving the t cell reponse

Question 30

what is gsea

Answer 30

-gene set enrichment analysis
-determines whether the members of one gene set are over represented in another gene sets

Question 31

How does flow cytometry work

Answer 31

-Flow cytometry [1] uses a fluidics system that feeds a stream of stained cells through a laser beam
-. “Staining”: the addition and binding of a fluorophore-conjugated antibody to a cellular component (often a protein on the cell surface).
-Fluoresence intensities of the antibodies bound per cell are determined by the flow cytometeric analysis. The fluoresence intensities of the antibodies bound per cell are
directly proportional to the expression levels of the proteins specifically recognized by the distinct antibodies

Question 32

strenghts of flow

Answer 32

-Absolute cell numbers of many distinct cells per tissues can be determined fast.
-Expression levels (through mean fluorescence intensities) of many distinct markers/molecules per cell can be distinguished of many cells rapidly and reliably

Question 33

weaknesses of flow

Answer 33

Digestion of tissues can be problematic and can change cell surface expression levels of molecules of interest and can alter fitness of cells.
-Expensive and antibodies/probes to detect markers need to be labelled to distinctfluorochromes

Question 34

how is intracellular staining done

Answer 34

Intracellular Stainings are done by fixing the cells with para(formaldehyde) to crosslink primary amino-termini of the cells. A specific buffer system is used to
permeabilize the cells to allow entry of antibodies to recognize the target of interest. It is important that isotype control antibodies are used for intracellular stainings.

Question 35

how does an elisa work

Answer 35

Enzyme-linked Immunosorbent assasy (ELISA) was used here, which requires 2 distinct
monoclonal antibodies that recognize 2 distinct epitopes of the protein dosed for (e.g. IL33). One antibody is used to capture the protein of interest in a plate-bound assay. The
second antibody detects (detection antibody) the protein of interest captured by the first
antibody; this detection antibody is labelled to an enzyme that allows a chromogen-based
readout – allowing directly proportional detection of the protein of interest.

Question 36

strenght of elisa

Answer 36

allows for abselute quantification of a protein of interest

Question 37

weaknesses of elisa

Answer 37

-does not allow single-cell analysis of a protein of interest.
-ELISA does not allows require tissue digestion and single cell analysis.

Question 38

cytof strenghts

Answer 38

-High Parameter Multiplexing: CyTOF allows for the simultaneous measurement of a large number of parameters (typically more than 40) on a single cell,

-Reduced fluorescence spillover
-reduced backround noise

-

Question 39

weaknesses iof cytof

Answer 39

-Cost: CyTOF instruments and consumables are expensive,
-Limited Sample Throughput: compared to flow
-Cell Viability: you kill the cells

Question 40

strenghts of cre lox

Answer 40

-Site-Specific Recombination: Cre-lox allows for precise, site-specific recombination

-Versatility: The Cre-lox system is versatile and can be applied in various organisms, including bacteria, yeast, plants, and animals

-Inducible Systems: Cre-lox can be combined with inducible promoters, allowing for temporal control over gene expression or deletion.

-Reversibility: The Cre-lox system allows for reversible genetic modifications.

Question 41

weaknesses of cre lox

Answer 41

-Off-Target Effects: Cre recombinase can sometimes mediate recombination at undesired off-target sites, leading to unintended genetic modifications.

Incomplete Recombination: In some cases, complete recombination may not occur, leading to the persistence of floxed alleles or the generation of mosaic animals with a mix of modified and unmodified cells.

Size Limitations: There are size limitations for the DNA fragments that can be efficiently recombined by the Cre-lox system. Large DNA fragments may have lower recombination efficiency.

Cre Toxicity: The expression of Cre recombinase itself can be toxic to cells, and high levels of Cre expression may result in non-specific recombination events or other cellular stresses.

Need for Breeding Schemes:

Question 42

how does rna seq work bulk

Answer 42

RNA is extracted from cells and then purified to mRNA by either poly- A selection, rRNA depletion, and/or size selection (electrophoresis). mRNA is frag- mented, adapter sequences are ligated to fragment ends, and a complementary DNA (cDNA) library is generated by reverse transcription. After amplification by polymerase chain reaction (PCR), the fragments are “read” using a next-generation sequencing plat- form such as those developed by Illumina, PacBio, or Oxford Nanopore Technologies.
-Alignment of reads to a reference genome. Regions where many reads pile up indicate regions that are more highly expressed. The expression level of each gene in a sample can be quantified using computational tools.

Question 43

strenghts of rna seq

Answer 43

Quantitative Measurement: RNA-Seq provides quantitative information about gene expression levels,

Genome-Wide Coverage: RNA-Seq can capture information from the entire transcriptome, providing a comprehensive view of gene expression across the entire genome.

Detection of Novel Transcripts: RNA-Seq can identify novel transcripts, splice variants, and non-coding RNAs that may not be represented in existing gene annotations.

Single-Nucleotide Resolution: RNA-Seq allows for the detection of single-nucleotide polymorphisms (SNPs) and mutations within transcribed regions, providing valuable information for genetic studies.

Question 44

weaknesses of rna seq

Answer 44

Cost: While the cost of RNA-Seq has decreased over time, it can still be relatively expensive, especially for large-scale studies involving many samples.

Sensitivity to RNA Integrity: The quality of RNA is critical for accurate results in RNA-Seq.

Difficulty in Detecting Low-Expressed Genes: Despite the wide dynamic range, RNA-Seq may struggle to accurately quantify very low-expressed genes due to limitations in library preparation and sequencing depth.

Inability to Distinguish Isoforms: In some cases, it may be challenging to accurately distinguish between highly similar isoforms or transcript variants, especially if they share common exons.

Question 45

how does scrna seq work

Answer 45

Recent methods use microfluidic encapsulation of cells into droplets with unique cellular “barcodes” and reagents for a reverse transcription reac- tion. After the reaction, cDNA from many cells can be mixed together for bulk-style sequencing since transcripts can be identified by their unique barcodes.

Question 46

strenghts of scrna seq

Answer 46

Cellular Heterogeneity: scRNA-Seq allows the study of gene expression at the single-cell level

Identification of Cell Types: scRNA-Seq facilitates the discovery and characterization of distinct cell types, subpopulations, and transitional states within complex tissues or samples.

Cell Trajectories and Dynamics: It enables the reconstruction of cell trajectories and the study of dynamic processes such as cell differentiation, development, and response to stimuli over time.

Rare Cell Detection: scRNA-Seq is sensitive enough to detect and characterize rare cell types or states that may be overshadowed in bulk RNA-Seq analyses.

Question 47

weaknesses of scrna seq

Answer 47

Data Complexity: scRNA-Seq data is inherently more complex than bulk RNA-Seq data, and the analysis can be challenging, requiring specialized computational methods for data processing, normalization, and interpretation.

High Cost per Cell: The cost per cell in scRNA-Seq experiments can be higher compared to bulk RNA-Seq, especially when analyzing a large number of cells.fa

Question 48

how does chip seq works

Answer 48

First, protein and DNA must be crosslinked to preserve interactions between them. Next, the crosslinked DNA is fragmented and added to magnetic beads coated with an antibody specific for the target protein. These precipitated complexes are purified by magnetic selection, and the DNA is finally eluted and sequenced.

Question 49

strenghts of chip seq

Answer 49

Genome-Wide Profiling: ChIP-Seq allows genome-wide profiling of protein-DNA interactions, providing insights into the binding locations of transcription factors, histone modifications, and other chromatin-associated proteins.

Identification of Regulatory Elements: as enhancers, promoters, and insulators, and understanding their roles in gene regulation.

Epigenetic Studies: ChIP-Seq is widely used in epigenetic studies to investigate histone modifications, DNA methylation, and chromatin accessibility, providing insights into the epigenetic regulation of gene expression.

Question 50

weaknesses of chip seq

Answer 50

Resolution Limitations: The resolution of ChIP-Seq is influenced by several factors, including the size of DNA fragments, sequencing depth, and the quality of antibodies used. Low resolution can make it challenging to precisely pinpoint binding sites, especially in regions with closely spaced regulatory elements.

Cross-Linking Artifacts: Cross-linking of proteins to DNA can introduce artifacts, leading to potential inaccuracies in the identification of binding sites.

Complex Data Analysis: ChIP-Seq data analysis can be complex, involving multiple steps such as quality control, read alignment, peak calling, and downstream interpretation. This complexity may require specialized bioinformatics expertise.

Lack of Functional Information: While ChIP-Seq provides information about binding locations, it does not directly provide functional information about the impact of protein binding on gene expression or cellular function.

Question 51

WHAT do the x and y axis mean in gsea

Answer 51

• The x-axis represents a ranked list of genes based on correlation with a given
  phenotype. The gene-ranking metric varies in different analyses.
• The y-axis represents the running enrichment score, which reflects the degree to which a gene set is overrepresented in the ranked list of genes. The points that appear before the highest peak contribute most to the total enrichment score.