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week 1 Flashcards

1
Q

What does biomarkers have great significance for?

A 
Prediction 
Diagnosis 
Monitoring 
Treatment 
And prognosis of many diseases
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2
Q

What are sampling types?

A 
Imaging 
Tissue + CSF
Blood
Breath
Saliva, semen urine, stool
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3
Q

Example of Imaging

A

CT and MRI

Can see brain injury

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4
Q

Example of blood

A

Brings all the goodies
Separated by blood brain barrier
Injured part of the cerebellum and/or brainstem

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5
Q

Example of breath

A

Used in lung cancer studies

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6
Q

Example of saliva, urine and stool

A

Saliva - useful biofluid to study AD
Stool is an interesting biosample
If it has bad bacteria - cause for abnormal brain function

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7
Q

Sampling: site of disease

A

The closer to the brain or spinal cord tissue, the more concentrated the analytes and the source of cause or effect of the disease

Accessibility is a big problem ethically
Taking brain samples can cause further damage

As we move away from CNS tissue, it makes the sampling a lot easier

Consequence: it is further away from the disease - analytes for study reduces as well

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8
Q

What are 3 types of sampling: tissue?

A

Fresh
Fresh-Frozen
Chemical Fixation

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9
Q

What are examples of fresh tissue and it’s storage properties

A

E.g. life cell imaging and cell culture

Store in media or buffer at 4 degree Celsius

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10
Q

What are pro and cons of fresh tissue

A

Pro: when live cells are required (e.g. cel culture, redox or metabolism studies)

Cons;
Easy degradation
Lack morphological detail
Potential biohazards

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11
Q

What are examples of fresh-frozen tissue?

A

Prevent tissue degradation
E.g. histology and biochemical studies
Store in liquid nitrogen/ -80 degrees

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12
Q

What are pros and cons of Fresh-frozen tissue?

A

Pros:
Long term storage of tissue
Better antigenicity preservation
Fast fixation method

Cons:
Lack morphological detail
Potential biohazard
Require specialised storage facilities

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13
Q

What are examples of chemical fixation tissue?

A

Maintain cellular structure

E.g. formalin-fixed paraffin embedded (FFPE)

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14
Q

What are pros and cons of chemical fixation tissue?

A

Pros:
Long term storage of tissue
Excellent tissue morphology
Store samples at room temperature

Cons:
Some epitopes are reduced/ damaged if fixed
(E.g. Leukocyte surface markers)
Researcher expose to fixative

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15
Q

Writing Ethics for using “brain from bin”

A 
70 page document that require:
General public and patient surveys 
Consent form
GP letters 
What you plan to do with it in detail 
Funding 
Reason for ethics
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16
Q

Formalin to immersion fix the tissue

A

Perfuse fix tissue with 4% paraformaldehyde
Paraformaldehyde is a colourless gas - irritating odour
Heat in water to form formaldehyde solution - equivalent to 10% formalin
Cross-link the primary amino acids in proteins with nearby nitrogen atoms in proteins and DNA

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17
Q

What is FFPE tissue?

A

Most widely used method for clinical sample preparation and archiving in
Hospitals
Tissue banks
Research labs

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18
Q

What is FFPE tissue used for?

A

Nucleic acid extraction

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19
Q

What is FFPE tissue not possible for?

A

Used in molecular analysis histologically

Over a billion tissue samples

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20
Q

What is nucleic acid?

A

Heavily modified

Trapped by extensive protein-nucleic acid and protein-protein cross linking

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21
Q

What can archived samples provide?

A

Wealth of information in retrospective molecular studies of diseases tissues?

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22
Q

How is PPFE tissue used?

A

Appropriate protease digestion e.g. trypsin

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23
Q

What can FFPE samples release?

A

Microgram amounts of DNA and RNA

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24
Q

What is the purified nucleic acid (fragmented) suitable for?

A

Variety of downstream genomic and gene expression analysis e.g.
Microarray
MicroRNA
Methylation profiling

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25
What biofluids to use?
Saliva Urine Blood CSF
26
Why is urine difficult to obtain?
Problems with water works
27
What is urine not subjected to?
``` Homeostatic mechanism Changes frequently and dramatically depends on Diet Fluid intake Dehydration. ```
28
What changes does urine accommodate that reflect status of body?
Pregnancy Aging Daily rhythms
29
When is urine monitored?
During the progression of many diseases
30
Why is the brain and urine not closely related?
Filtering effects of BBB and kidneys
31
Why is saliva a good biofluid source?
Study of diagnostic biomarkers of AD using metabolomics Ease and convenience of collecting saliva
32
What has most brain disease studies focused on?
CSF | Blood
33
Urine had been used as a biofluid for the study of what brain disorders?
Schizophrenia e.g. glutamic acid, glucosamine Parkinson’s disease e.g. histidine, dihydrocortisol Stroke e.g. glycine and hippurate
34
How is blood easily and routinely extracted by?
Venipuncture | Finger prick
35
How many pints of blood does adults have in body?
10 UK pints
36
What are the sampling volumesv
1 pint (568 ml) every 56 days (healthy and fit adult) 10 ml every day 1ml regularly Dried blood spots
37
Why can blood be considered as tissue?
Contains millions of cells Perform similar functions Carry out transport functions and protect the mammal disease
38
What is blood tissue made up of?
Fluid called plasma Red and white blood cells float with this plasma Glucose and amino acids molecules are also found in plasma - dissolved form
39
What is functions of blood?
Transport medium Carries substances around the body Substances; dissolved food, oxygen and plasma proteins Carries waste substances to excretion organs: lungs and kidneys Help with heat distribution from internal organs to fingers and toes Homeostasis WBC attack foreign particles and germs
40
What are the 2 most common blood sampling tubes?
Purple/lavender top | Red top
41
What does purple top contain?
EDTA additive | Used for: haematology and transfusion services
42
What does red top contain?
Clot activator Specimen clots in 60 Mins Used for: serum chemistry and serology assay Invert tubes 5 times
43
What are the variables that may impact analytic outcome?
``` Type of additive in the blood collection tube Sample processing times or temperatures Hemolysis of sample Sample storage parameters The number of freeze-thaw cycles ```
44
How can hemolysid (pink to red tinge in the sample) be prevented?
Careful handling technique Optimum needle size Proper handling of the tubes Pipette technique to reduce incidence of hemolysis
45
What is serum?
Liquid portion of the blood without cells and clotting factors Contains proteins and other molecules that represent the whole body system Cells and clotting factors must be removed from blood sample by allowing time for clot to form 30-60 min at room temperature for a clot to form and longer
46
What is plasma?
Includes cellular material - provide different analytes Plasma collection tube contain different anticoagulant Can be used in proteomics and genomic analysis
47
Plasma
Liquid part of blood With clotting factors Pro: fibrinogen present in plasma Can be centrifuge immediately after gently 8-10 inversions Cons: anticoagulant required
48
What is serum?
Liquid part of blood without clotting factors Pros: anticoagulant not required Cons: fibrinogen absent in plasma Need standing time before centrifuge
49
Why is hemolysis a problem?
Release proteins from cells into fluid sections
50
What is cerebrospinal fluid ?
Clear and colourless liquid that surrounds brain and spinal cord
51
What are the 3 functions of CSF?
Mechanical and supportive Protective Metabolic
52
Mechanical and supportive
Suspend in fluid to prevent damage by its own weight | The brain is immersed in CSF causing a reduction of its real weight to an equivalent about 25g
53
Protective
Acts as a shock absorber Projects brain from a sudden pressure or temperature changes and the components of immune system present in this fluid protect against various pathogens
54
Metabolic
Liquor helps maintain correct composition of environment surrounding nervous tissue cells Supply of nutrients and disposal of metabolic waste products Form a medium through which diffusion of a various signal molecules takes place
55
What is blotting (Southern, Western, Northern)?
Transfer of DNA, RNA, protein onto a gel separated by molecular size Transferred to membrane for straining and identification
56
Southern
DNA blot
57
Northern
RNA blot
58
Eastern
Protein with PTM* Blot
59
Western blot
Pinpoint specific protein in a given sample Ability of an enzyme or fluorescently-labelled antibody to bind to its specific antigen Protein detection may be direct or indirect Latter uses a labelled secondary antibody directly against primary
60
What are 3 step process for Western blot?
Gel electrophoresis Membrane blotting Probing with antibodies
61
What are advantages of western blot?
Sensitivity | Specificity
62
Sensitivity of western blot
Detect as little as 0.1 nanogram of protein in a sample Early diagnostic tool Greater sensitivity means fewer antibodies are needed for testing Cuts down lab cost significantly
63
Specificity of western blot
Serves as detection as bands formed in gel gives clue about size of protein of interest Specificity of antibody-antigen interaction
64
What are disadvantages of western blot?
Prone to false/subjective results | High cost and technical demand
65
Prone to false/subjective results
Produce incorrect results False-positive - antibody reacts with non-intended protein False-negative - lather proteins are not given sufficient time to transfer properly to the membrane Improper blotting / faded or multiple bands Make test results subject to interpretation of technicians
66
High cost and technical demand
Requires precision in every step for proper identification Equipment are too expensive for average microbiology unit
67
SDS protocol
Get plasma sample Dilute Add SDS SDS puts negative charge onto protein and unravels Boil for 5 minutes and stick on ice Separate by size and put ladders of particular size to identify
68
What is ELISA
Plate-based technique - detect and quantify substances such as proteins, antibodies and hormones Assess conjugated enzyme activity via incubation. With a substrate to produce measurable produce Highly specific for antibody-antigen interaction Performed in a 96-well polystyrene plate
69
What are the most commonly used enzyme labels?
Horseradish peroxidase | Alkaline phosphatase
70
What is direct detection method
Use labelled primary antibodies that react directly with antigen Antigen is directly immobilised in assay plate Not widely used
71
What is indirect detection method
Use a labelled secondary antibody for detection Most popular format Secondary antibody has specificity for primary antibody
72
What are advantages for direct ELISA detection?
Quick - one antibody, fewer steps | Cross-reactivity of secondary antibody is eliminated
73
What are disadvantages of direct ELISA detection?
Immunoreactivity of primary antibody is affected by labelling with enzymes Labelling primary antibody for each specific ELISA system is time-consuming and expensive
74
What is gel-based proteomics?
Protein separated by 1-DE or 2-DE Enzyme digestion to form peptides Mass spectrometry analysis Database research
75
What is shot-gun proteomics
Enzyme digestion of protein mixture to form peptides Mass spectrometry analysis Database research
76
What is pros of 2-DE electrophoresis
Common method of protein identification using molecular size and PH Separates several thousands of protein on 1 gel Visualise proteins using silver or Coomassie Brilliant Blue standing to identify proteins that have different expression level
77
What are cons of 2-DE electrophoresis ?
Time consuming Labour intensive Restricted to detect denatured proteins between 10-209kDA at PH 3.5-11.5 Ineffective to distinguish low-abundant proteins PTM and Isoform information is lost
78
SYBR green dye method
Excessive SYBR green fluorescent dye is added into PCR reaction system SYBR green dye bind specifically to double-stranded DNA can emit fluorescence signal Allows DNA concentration to be quantified
79
Understanding qPCR results
Gene expression - qPCR will tell you how much of a specific MRNA there is in your sample
80
How do you do qPCR
Amplify a small region of MRNA with Oligos and fluorescent probe
81
What does qPCR machine measure?
Intensity of fluorescence emitted by probe at each cycle
82
What does qPCR have,
Exponential phase followed by a plateau phase
83
What does CT represent?
Basic results of the qPCR experience | Exponential phase - curve is linear
84
Where is threshold placed?
Linear phase
85
How is CT measured?
Where qPCR curve crosses the threshold
86
What is threshold different for?
For every qPCR assay
87
What is the principle of qPCR based on?
At each PCR cycle, the number of PCR product doubles
88
CT
Value that will be used for analysis
89
The higher the CT
The less MRNA defected
90
If the CT has a small value (10-15)
The gene is highly expressed
91
Sequencing: WGS
Myers proposed the idea of breaking many copies of entire genome into different sized pieces and sequencing those pieces in a special way
92
What does a huge percentage of human genome consist of?
Repeated non-coding sequences dispersed throughout the genome These regions include LINES and minisatellite are
93
What did Myers propose?
Entire genome be broken up into 2000, 10000, 50000 base pairs long
94
WES
Aid diagnosis of rare neurological disorders in pediatric population 1. Collect blood 2. Extract and fragment DNA 3. Capture exome DNS with probes 4. Recover only exome DNA fragments 5. Sequence on next-generation platform
95
Pros for WGS vs WES
Allow the study of a single-nucleotide variant More reliable sequencing coverage Uniformity of coverage is superior Do not require PCR amplification Species not an issue but WES is limited to humans due to probe production
96
Cons of WGS and WES
Expensive cost in sequencing Storage of data and bioinformatic analysis Large days require large computer power And expert in bioinformatic analysis
97
Next generation sequencing (NGS)
Sequence DNA and RNA much more quickly and cheaply Revolutionised study of genomics and molecular biology
98
What is 4 basic steps of ILLUMINA NGS
1. Library preparation 2. Cluster Generation 3. Sequencing 4. Data analysis
99
Library preparation
Sequencing library is prepared by random fragmentation of DNA or cDNA sample Followed by 5’ and 3’ adaptor ligation
100
Cluster Generation
Library loaded into a flow cell - fragments captured on surface - bound Oligos complementary to library adaptors Each fragment amplified into distinct, clonal clusters through bridge amplification The templates are ready for sequencing
101
Sequencing
Reversible terminator method that detect single bases as they are incorporated into DNA template strand
102
Data analysis
Newly identified sequence reads are aligned to a reference genome
103
Microarray
High-throughput and versatile technology - parallel gene expression analysis Orderly arrangement of thousands of identified sequences genes printed on a solid support
104
Mass spectrometry
Molecular fragments are ionised by a steam of electrons Accelerated by electrical field through a deflector magnet The lower the mass of ions, the more they are deflected by magnet

markers xxx (11 decks)

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