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Techniques Flashcards

1
Q

What is an oral gavage?

A

Using a needle to put liquid straight into the stomach from the mouth

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2
Q

Advantages of an oral gavage?

A

Precise oral dosing
Accurate and reliable method

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3
Q

Disadvantage of an oral gavage?

A

Increase stress
Aspiration
Esophageal trauma

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4
Q

What are alternative to a gavage?

A

Allowing the animal to drink from a syringe
Eat it in food

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5
Q

How do you interpret a western blot?

A

If the line is really thick it could be overloaded and therefore not interpretable

You look at size and darkness of band shows how much protein there is

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6
Q

How many sections should you take in a serial section?

A

At least 2

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7
Q

How many mice is the average number of mice to use in an experiment?

A

Around 10

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8
Q

When doing immunohistochemistry should you have both positive and negative controls?

A

Yes

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9
Q

When answering the bullets points what must you remember to do?

A

Say WHY and use example from the paper you are critically analysing

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10
Q

Benefits of mouse models?

A

Lots as they have a short generation span
Relatively small

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11
Q

What does it mean if something has a positive correlation?

A

As one goes up so to does the other

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12
Q

What does it mean by a negative correlation?

A

As one goes down so to does the other

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13
Q

What is an inverse correlation?

A

As one thing goes up the other goes down or vice versa - they move in opposite directions

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14
Q
A
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15
Q

What does gel electrophoresis do?

A

Separates DNA based on mass

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16
Q

What are the pros of gel electrophoresis?

A

Small quantities needed
Quantitative and qualitative
Clear band patterns

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17
Q

What are cons of gel electrophoresis?

A

Time consuming
Costly
Can lead to artifacts

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18
Q

What is the best technique for splitting dna?

A

Gel electrophoresis

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19
Q

What is the critical analysis for a gel electrophoresis?

A

Is there a ladder?
Is there smearing? - this means overloading
Unexpected band?
Is there multiple bands? - if so this means more than one dna fragment

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20
Q

What is a cell culture?

A

When cells are removed to examine

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21
Q

What are advantages of cell cultures?

A

Controlled experiment
Constant results

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22
Q

What are cons of a cell culture?

A

Potential contamination
In vivo conditions not met

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23
Q

What does a PCR or rPCR do?

A

Amplifies dna or rna

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24
Q

What are advantages of a PCR?

A

Fast way to sample dna/rna

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25
What are cons of a PCR?
Low specificity
26
What things to consider when critically analysing PCR results?
Has this been ran multiple times and if so are results consistent? Are there alternative explanations for these results?
27
What is a Q-PCR/Q-Rt-PCR
Quantitative PCR for dna and rna
28
What is the difference between a qpcr and a PCR?
QPCR gives quantitative results as is more accurate
29
Pros of a QPCR?
High specificity and sensitivit
30
Cons of a QPCR?
Cost Potential for false results
31
How to critically analyse a QPCR?
Similar to a PCR
32
What is gene cloning?
When genes are cloned to get larger quantities of protein
33
What are pros of gene cloning?
Large quantities of proteins generated DNA sequencing can be done
34
What are cons of gene cloning?
Ethics Technical difficulties Cost
35
What is a western blot?
Helps detect proteins
36
What are pros of a western blot?
Detect wide range of proteins Specific and sensitive
37
What are cons for a western blot?
Semi quantitive
38
What are some critical analysis points for a western blot?
Has a t-test been done Is there smearing? Ladder? House keeping gene? You want it to be quantitative to analyse
39
What does SDS-PAGE do?
Visualise proteins by making them -ve charged and they travel towards the positive anode.
40
What are pros of SDS-PAGE?
High resolution, Good Reproducibility High Sensitivity
41
What are cons of SDS-PAGE?
Can denature proteins Unable to separate out similar molecular weights
42
What things to think about when critically analysing SDS-PAGE?
Is there a ladder? Protein smearing or broken lines = indicates protein degradation. Can't distinguish between similar molecular weighted items.
43
What is an ELISA?
ELISA's are done to analyse concentrations of hormones
44
Pros of an ELISA?
Simple procedure High specificity and sensitivity High efficacy
45
Cons of an ELISA?
False positives are common Time consuming and labour extensive Antibody stability is a concern
46
Critical analysis points of an ELISA?
Has it been replicated? Positive and negative controls?
47
What is sanger sequencing?
Determines the nucleic acid sequence
48
What are pros of sanger sequencing?
High accuracy
49
What are cons of sanger sequencing?
Hard to look at large deletions
50
Critical analysis points for sanger sequencing?
Look for sharp peaks, Be aware of back ground peaks e.g. background info
51
What does RNA in situ hybridisation do?
Looks at specific nucleic acid sequences - DNA and RNA
52
Pros of RNA in situ hybridisation?
High specificity and sensitivity Versatile
53
Cons of RNA in situ hybridisaton?
Artifacts and misinterpretation Limits of sensitivity Technical Challenges
54
Critical Analysis of RNA in situ hybridisation?
Controls need to be used, Careful background staining Needs quantification Clear labelling
55
Why do we use genetically modified animals?
The find out what a gene does Model Human disease
56
What is transgenesis?
The process of introducing a foreign gene into an organisms genome
57
What are the two different ways to target genes?
Knock out (specific genes) and knock ins (specific genes)
58
Pros of transgenesis?
Fast and inexpensive
59
Cons of transgenesis?
Aberrant transgene expression Insertional mutagenesis
60
Pros of gene targeting?
More control
61
Cons of gene targeting?
Cost and large time frame
62
If a study uses transgenesis what can you suggest instead?
Gene targeting as it is more precise and easier to control
63
What are the different type of genetically modified mice?
Reporter mice Knockout - permanent deletion can be embryo lethal Conditional Knockout - Cre/loxP CRISPR
64
What is mechanical dissociation?
cutting, crushing, scraping, pipetting etc, to get loosely associated samples
65
Pros of mechanical dissociation?
Fastest and simplest
66
Cons of mechanical dissociation?
Inconsistent results between users Affect cell viability if to harsh
67
What is enzymatic dissociation?
Target specific protein and used for more compact tissue
68
Pros of enzymatic dissociation?
Efficient
69
cons of enzymatic dissociation?
Longer than mechanical and can modify proteins on the surface of the cell
70
What is chemical dissociation?
targets cations which hold together intracellular bonds
71
Cons of chemical dissociation?
Can take a long time
72
Do they combine the different types of dissociation?
Yes
73
What is MACS used for?
Cell sorting
74
How does MACS work?
Magnetic beads coated in with antibodies target specific cells and then pull them to the magnetised sides allowing not magnetic beaded cells to pass through.
75
Pros of MACS
Fast
76
Cons of MACS?
Harsh method that can be harmful to cells Costly
77
How to do BACS?
Cell separated using buoyant microbubbles.
78
Pros of BACS?
Gentle, less harsh
79
What is FACS?
Uses fluorescent labelling to target and isolate groups of cells
80
Pros of FACS?
high-purity cell isolation
81
Cons of FACS?
Can cause damage to cells
82
Positives of in vivo models?
Effect of offspring Real Life Downstream/long term effects
83
Cons of in vivo models?
Expense Ethical concerns Less easy to control conditions Harder to get humans involved
84
Pros of in vitro models?
Less animals Real time observation Patient specific
85
Cons of in vitro?
Longer to establish method Antibiotic interference No hormones
86
What are the benefits of using large animals?
Lots of clinical material Foetal development is more similar to humans esp sheep who have one or two young, You can do multiple tests on an animal
87
Cons of using large animals?
Expense Resources Species-specific associations
88
What are microarrays?
Provide information about the transcriptome
89
Cons of microarrays?
Requires prior knowledge of the genome Sensitive to cross hybridisation Low signal to noise ratio limits
90
What is next generation (Deep) Sequencing?
Looks at the human genome
91
Things to remember to include in a graphical abstract:
The different groups Method Results
92
Should there be lots of writing in a graphical abstract?
No
93
Can you use colour in a graphical abstract?
Yes
94
When doing the review how should you start?
Summary of paper

Synoptic Exam (29 decks)

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