week 1 Flashcards
What does biomarkers have great significance for?
Prediction Diagnosis Monitoring Treatment And prognosis of many diseases
What are sampling types?
Imaging Tissue + CSF Blood Breath Saliva, semen urine, stool
Example of Imaging
CT and MRI
Can see brain injury
Example of blood
Brings all the goodies
Separated by blood brain barrier
Injured part of the cerebellum and/or brainstem
Example of breath
Used in lung cancer studies
Example of saliva, urine and stool
Saliva - useful biofluid to study AD
Stool is an interesting biosample
If it has bad bacteria - cause for abnormal brain function
Sampling: site of disease
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
What are 3 types of sampling: tissue?
Fresh
Fresh-Frozen
Chemical Fixation
What are examples of fresh tissue and it’s storage properties
E.g. life cell imaging and cell culture
Store in media or buffer at 4 degree Celsius
What are pro and cons of fresh tissue
Pro: when live cells are required (e.g. cel culture, redox or metabolism studies)
Cons;
Easy degradation
Lack morphological detail
Potential biohazards
What are examples of fresh-frozen tissue?
Prevent tissue degradation
E.g. histology and biochemical studies
Store in liquid nitrogen/ -80 degrees
What are pros and cons of Fresh-frozen tissue?
Pros:
Long term storage of tissue
Better antigenicity preservation
Fast fixation method
Cons:
Lack morphological detail
Potential biohazard
Require specialised storage facilities
What are examples of chemical fixation tissue?
Maintain cellular structure
E.g. formalin-fixed paraffin embedded (FFPE)
What are pros and cons of chemical fixation tissue?
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
Writing Ethics for using “brain from bin”
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
Formalin to immersion fix the tissue
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
What is FFPE tissue?
Most widely used method for clinical sample preparation and archiving in
Hospitals
Tissue banks
Research labs
What is FFPE tissue used for?
Nucleic acid extraction
What is FFPE tissue not possible for?
Used in molecular analysis histologically
Over a billion tissue samples
What is nucleic acid?
Heavily modified
Trapped by extensive protein-nucleic acid and protein-protein cross linking
What can archived samples provide?
Wealth of information in retrospective molecular studies of diseases tissues?
How is PPFE tissue used?
Appropriate protease digestion e.g. trypsin
What can FFPE samples release?
Microgram amounts of DNA and RNA
What is the purified nucleic acid (fragmented) suitable for?
Variety of downstream genomic and gene expression analysis e.g.
Microarray
MicroRNA
Methylation profiling
What biofluids to use?
Saliva
Urine
Blood
CSF
Why is urine difficult to obtain?
Problems with water works
What is urine not subjected to?
Homeostatic mechanism Changes frequently and dramatically depends on Diet Fluid intake Dehydration.
What changes does urine accommodate that reflect status of body?
Pregnancy
Aging
Daily rhythms
When is urine monitored?
During the progression of many diseases
Why is the brain and urine not closely related?
Filtering effects of BBB and kidneys
Why is saliva a good biofluid source?
Study of diagnostic biomarkers of AD using metabolomics
Ease and convenience of collecting saliva
What has most brain disease studies focused on?
CSF
Blood
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
How is blood easily and routinely extracted by?
Venipuncture
Finger prick
How many pints of blood does adults have in body?
10 UK pints
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
Why can blood be considered as tissue?
Contains millions of cells
Perform similar functions
Carry out transport functions and protect the mammal disease
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
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
What are the 2 most common blood sampling tubes?
Purple/lavender top
Red top
What does purple top contain?
EDTA additive
Used for: haematology and transfusion services
What does red top contain?
Clot activator
Specimen clots in 60 Mins
Used for: serum chemistry and serology assay
Invert tubes 5 times
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
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
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
What is plasma?
Includes cellular material - provide different analytes
Plasma collection tube contain different anticoagulant
Can be used in proteomics and genomic analysis
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
What is serum?
Liquid part of blood without clotting factors
Pros: anticoagulant not required
Cons: fibrinogen absent in plasma
Need standing time before centrifuge
Why is hemolysis a problem?
Release proteins from cells into fluid sections
What is cerebrospinal fluid ?
Clear and colourless liquid that surrounds brain and spinal cord
What are the 3 functions of CSF?
Mechanical and supportive
Protective
Metabolic
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
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
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
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
Southern
DNA blot
Northern
RNA blot
Eastern
Protein with PTM* Blot
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
What are 3 step process for Western blot?
Gel electrophoresis
Membrane blotting
Probing with antibodies
What are advantages of western blot?
Sensitivity
Specificity
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
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
What are disadvantages of western blot?
Prone to false/subjective results
High cost and technical demand
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
High cost and technical demand
Requires precision in every step for proper identification
Equipment are too expensive for average microbiology unit
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
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
What are the most commonly used enzyme labels?
Horseradish peroxidase
Alkaline phosphatase
What is direct detection method
Use labelled primary antibodies that react directly with antigen
Antigen is directly immobilised in assay plate
Not widely used
What is indirect detection method
Use a labelled secondary antibody for detection
Most popular format
Secondary antibody has specificity for primary antibody
What are advantages for direct ELISA detection?
Quick - one antibody, fewer steps
Cross-reactivity of secondary antibody is eliminated
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
What is gel-based proteomics?
Protein separated by 1-DE or 2-DE
Enzyme digestion to form peptides
Mass spectrometry analysis
Database research
What is shot-gun proteomics
Enzyme digestion of protein mixture to form peptides
Mass spectrometry analysis
Database research
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
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
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
Understanding qPCR results
Gene expression - qPCR will tell you how much of a specific MRNA there is in your sample
How do you do qPCR
Amplify a small region of MRNA with Oligos and fluorescent probe
What does qPCR machine measure?
Intensity of fluorescence emitted by probe at each cycle
What does qPCR have,
Exponential phase followed by a plateau phase
What does CT represent?
Basic results of the qPCR experience
Exponential phase - curve is linear
Where is threshold placed?
Linear phase
How is CT measured?
Where qPCR curve crosses the threshold
What is threshold different for?
For every qPCR assay
What is the principle of qPCR based on?
At each PCR cycle, the number of PCR product doubles
CT
Value that will be used for analysis
The higher the CT
The less MRNA defected
If the CT has a small value (10-15)
The gene is highly expressed
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
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
What did Myers propose?
Entire genome be broken up into 2000, 10000, 50000 base pairs long
WES
Aid diagnosis of rare neurological disorders in pediatric population
- Collect blood
- Extract and fragment DNA
- Capture exome DNS with probes
- Recover only exome DNA fragments
- Sequence on next-generation platform
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
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
Next generation sequencing (NGS)
Sequence DNA and RNA much more quickly and cheaply
Revolutionised study of genomics and molecular biology
What is 4 basic steps of ILLUMINA NGS
- Library preparation
- Cluster Generation
- Sequencing
- Data analysis
Library preparation
Sequencing library is prepared by random fragmentation of DNA or cDNA sample
Followed by 5’ and 3’ adaptor ligation
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
Sequencing
Reversible terminator method that detect single bases as they are incorporated into DNA template strand
Data analysis
Newly identified sequence reads are aligned to a reference genome
Microarray
High-throughput and versatile technology - parallel gene expression analysis
Orderly arrangement of thousands of identified sequences genes printed on a solid support
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
