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markers xxx > week 1 > Flashcards

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
Q

What biofluids to use?

A

Saliva
Urine
Blood
CSF

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26
Q

Why is urine difficult to obtain?

A

Problems with water works

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27
Q

What is urine not subjected to?

A 
Homeostatic mechanism 
Changes frequently and dramatically depends on 
Diet 
Fluid intake 
Dehydration.
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28
Q

What changes does urine accommodate that reflect status of body?

A

Pregnancy
Aging
Daily rhythms

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29
Q

When is urine monitored?

A

During the progression of many diseases

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30
Q

Why is the brain and urine not closely related?

A

Filtering effects of BBB and kidneys

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31
Q

Why is saliva a good biofluid source?

A

Study of diagnostic biomarkers of AD using metabolomics

Ease and convenience of collecting saliva

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32
Q

What has most brain disease studies focused on?

A

CSF

Blood

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33
Q

Urine had been used as a biofluid for the study of what brain disorders?

A

Schizophrenia e.g. glutamic acid, glucosamine

Parkinson’s disease e.g. histidine, dihydrocortisol

Stroke e.g. glycine and hippurate

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34
Q

How is blood easily and routinely extracted by?

A

Venipuncture

Finger prick

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35
Q

How many pints of blood does adults have in body?

A

10 UK pints

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36
Q

What are the sampling volumesv

A

1 pint (568 ml) every 56 days (healthy and fit adult)

10 ml every day

1ml regularly

Dried blood spots

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37
Q

Why can blood be considered as tissue?

A

Contains millions of cells
Perform similar functions
Carry out transport functions and protect the mammal disease

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38
Q

What is blood tissue made up of?

A

Fluid called plasma
Red and white blood cells float with this plasma
Glucose and amino acids molecules are also found in plasma - dissolved form

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39
Q

What is functions of blood?

A

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

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40
Q

What are the 2 most common blood sampling tubes?

A

Purple/lavender top

Red top

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41
Q

What does purple top contain?

A

EDTA additive

Used for: haematology and transfusion services

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42
Q

What does red top contain?

A

Clot activator
Specimen clots in 60 Mins
Used for: serum chemistry and serology assay
Invert tubes 5 times

43
Q

What are the variables that may impact analytic outcome?

A 
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
Q

How can hemolysid (pink to red tinge in the sample) be prevented?

A

Careful handling technique
Optimum needle size
Proper handling of the tubes
Pipette technique to reduce incidence of hemolysis

45
Q

What is serum?

A

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
Q

What is plasma?

A

Includes cellular material - provide different analytes
Plasma collection tube contain different anticoagulant
Can be used in proteomics and genomic analysis

47
Q

Plasma

A

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
Q

What is serum?

A

Liquid part of blood without clotting factors

Pros: anticoagulant not required

Cons: fibrinogen absent in plasma
Need standing time before centrifuge

49
Q

Why is hemolysis a problem?

A

Release proteins from cells into fluid sections

50
Q

What is cerebrospinal fluid ?

A

Clear and colourless liquid that surrounds brain and spinal cord

51
Q

What are the 3 functions of CSF?

A

Mechanical and supportive
Protective
Metabolic

52
Q

Mechanical and supportive

A

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
Q

Protective

A

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
Q

Metabolic

A

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
Q

What is blotting (Southern, Western, Northern)?

A

Transfer of DNA, RNA, protein onto a gel separated by molecular size
Transferred to membrane for straining and identification

56
Q

Southern

A

DNA blot

57
Q

Northern

A

RNA blot

58
Q

Eastern

A

Protein with PTM* Blot

59
Q

Western blot

A

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
Q

What are 3 step process for Western blot?

A

Gel electrophoresis
Membrane blotting
Probing with antibodies

61
Q

What are advantages of western blot?

A

Sensitivity

Specificity

62
Q

Sensitivity of western blot

A

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
Q

Specificity of western blot

A

Serves as detection as bands formed in gel gives clue about size of protein of interest

Specificity of antibody-antigen interaction

64
Q

What are disadvantages of western blot?

A

Prone to false/subjective results

High cost and technical demand

65
Q

Prone to false/subjective results

A

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
Q

High cost and technical demand

A

Requires precision in every step for proper identification

Equipment are too expensive for average microbiology unit

67
Q

SDS protocol

A

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
Q

What is ELISA

A

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
Q

What are the most commonly used enzyme labels?

A

Horseradish peroxidase

Alkaline phosphatase

70
Q

What is direct detection method

A

Use labelled primary antibodies that react directly with antigen

Antigen is directly immobilised in assay plate

Not widely used

71
Q

What is indirect detection method

A

Use a labelled secondary antibody for detection

Most popular format

Secondary antibody has specificity for primary antibody

72
Q

What are advantages for direct ELISA detection?

A

Quick - one antibody, fewer steps

Cross-reactivity of secondary antibody is eliminated

73
Q

What are disadvantages of direct ELISA detection?

A

Immunoreactivity of primary antibody is affected by labelling with enzymes

Labelling primary antibody for each specific ELISA system is time-consuming and expensive

74
Q

What is gel-based proteomics?

A

Protein separated by 1-DE or 2-DE
Enzyme digestion to form peptides
Mass spectrometry analysis
Database research

75
Q

What is shot-gun proteomics

A

Enzyme digestion of protein mixture to form peptides
Mass spectrometry analysis
Database research

76
Q

What is pros of 2-DE electrophoresis

A

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
Q

What are cons of 2-DE electrophoresis ?

A

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
Q

SYBR green dye method

A

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
Q

Understanding qPCR results

A

Gene expression - qPCR will tell you how much of a specific MRNA there is in your sample

80
Q

How do you do qPCR

A

Amplify a small region of MRNA with Oligos and fluorescent probe

81
Q

What does qPCR machine measure?

A

Intensity of fluorescence emitted by probe at each cycle

82
Q

What does qPCR have,

A

Exponential phase followed by a plateau phase

83
Q

What does CT represent?

A

Basic results of the qPCR experience

Exponential phase - curve is linear

84
Q

Where is threshold placed?

A

Linear phase

85
Q

How is CT measured?

A

Where qPCR curve crosses the threshold

86
Q

What is threshold different for?

A

For every qPCR assay

87
Q

What is the principle of qPCR based on?

A

At each PCR cycle, the number of PCR product doubles

88
Q

CT

A

Value that will be used for analysis

89
Q

The higher the CT

A

The less MRNA defected

90
Q

If the CT has a small value (10-15)

A

The gene is highly expressed

91
Q

Sequencing: WGS

A

Myers proposed the idea of breaking many copies of entire genome into different sized pieces and sequencing those pieces in a special way

92
Q

What does a huge percentage of human genome consist of?

A

Repeated non-coding sequences dispersed throughout the genome

These regions include LINES and minisatellite are

93
Q

What did Myers propose?

A

Entire genome be broken up into 2000, 10000, 50000 base pairs long

94
Q

WES

A

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
Q

Pros for WGS vs WES

A

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
Q

Cons of WGS and WES

A

Expensive cost in sequencing
Storage of data and bioinformatic analysis

Large days require large computer power
And expert in bioinformatic analysis

97
Q

Next generation sequencing (NGS)

A

Sequence DNA and RNA much more quickly and cheaply

Revolutionised study of genomics and molecular biology

98
Q

What is 4 basic steps of ILLUMINA NGS

A
  1. Library preparation
  2. Cluster Generation
  3. Sequencing
  4. Data analysis
99
Q

Library preparation

A

Sequencing library is prepared by random fragmentation of DNA or cDNA sample

Followed by 5’ and 3’ adaptor ligation

100
Q

Cluster Generation

A

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
Q

Sequencing

A

Reversible terminator method that detect single bases as they are incorporated into DNA template strand

102
Q

Data analysis

A

Newly identified sequence reads are aligned to a reference genome

103
Q

Microarray

A

High-throughput and versatile technology - parallel gene expression analysis

Orderly arrangement of thousands of identified sequences genes printed on a solid support

104
Q

Mass spectrometry

A

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

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