lab research Flashcards
why is laboratory-based biomedical research important?
- not all questions can be answered using humans
5 things that laboratory-based biomedical research includes?
1.exploring new phenomena
2.understanding diseases mechanisms
3.testing new hypotheses
4.screening for inhibitors
5.identifying targets/candidates
lab research vs field research
-controlled- variables can be —well controlled
reproducibility
-clear conclusions
-artificial/ non physiological settings
-may not translate well to humans
-expensive
8 steps of process of lab research
1.defining research Q
2.defines aims of project
3.design and plan experiments
4.order reagents and equipment
5.tool or method development
6.tool or method development
7.conduct experiments
8.data analysis
9.conclusions
what 2 process used for DNA
PCR, genome sequencing
what 2 process is used for
RT-PCR , RNAsea
what 3 process used for protein?
1.PAGE
2.proteotamics
3.immunblotting
DNA extraction
methods involve disruption of cells using lysis regents and subsequent treatment with different chemicals remove other biomolecules
common method in DNA extraction
organic extraction using phenomenal and chloroform, both mixtures denature and precipitate out portions and treatments removed the cellular RNA
2 Specialised protocols used for extraction of high quality DNA from clinical samples
1.blood
2.feaces
Polymerase chain reaction(PCR)
new development and modification to increase accuracy and efficiency an enzyme
6 PCR features
1.cloning
2.disease diagnosis
3.detection of infectious agents
4.forensics
5.evolutionary biology
6.Archeology-ancient DNA anaylsis
Genome sequencing
DNA sequence of the genome of an organism
4 basics of whole genome(shotgun) sequencing
-high quality genomic DNA
-sheared to small fragments
-tagged with adapted molecules and amplified
sequenced in a sequencing machine such as illuminating
6 applications of illumination sequencing
1.disease diagnosis/predication in clinical medicine
2.prenatal, newborn screening
3.infectious disease epidemiology
4.bacterial, viral evolution studies
5.human population history studies
6.personalised medicine
RNA(reverse transcriptionPCR)
synthesis of complementary DNA from RNA
What does reverse transcriptase occur in?
RNA viruses(retroviruses)-mediates conversion from RNA to DNA
what happens in RT-PCR?
reverse transcription is coupled with PCR
3 Applications of RT-PCR
1.generation of cDNA for cloning and cDNA libraries
2.recombinant protein expression
3.key to several global transcriptomic methodologies e.g. RNA seq
3 things RNAseq does?
1.sequencing all transcripts in a cell
2.global view/snapshot of which genes are on/off at a time
3.quantitate gene expression
is the PCR method quantitative or qualitative?
quanitative-is more sensitive and specific
5 applications of PCR
1.disease diagnosis-quantitate specific transcripts
2.measure microbial load
3.gene expression studies
4.SNP analysis
5.genotyping
Polacrylamide gel electrophoresis(PAGE)
basic method for analysing cellular protein
4 steps of process in SDS-PAGE
1.denaturing proteins using detergent sodium dodecyl sulphate(SDS)
2.elimates structure and charge of protiens
3.separation based only on molecular weight of polypeptides
4.widely used to separate proteins and visualise protein profiles
3 things western-blotting(immunoblotting) does?
1.protiens on membranes are detected using specific antibodies
2.high sensitivity detects low amount of proteins
3.detect specific proteins and quantitate proteins
what does ELISA(enzyme-linked immunosorbent assay) measure?
antibodies, or specific protiens from biological samples
3 applications of ELISA
1.HIV detection
2.measuring cytokines
3.pregnancy test
in Proteomics what is proteome?
set of proteins produced by a cell, system or organism under a set of conditions
what is proteome used to study?
protein production
stability
control
protein modifications
protein interactions
protein movement across sub cellular compartments
in vitro cellular models
-cell lines or primary cells
in vitro cellular models, what are primary cells derived from
tissues
in vitro cellular models, what are cell lines?
cells are transformed to allow continuous propagation
3 things in vitro cellular modals do?
1.genetically manipulate
2.known out genes
3.visualise proteins(florescent labelled)
what does in vitro cellular assays study?
human responses
2 functions of in vitro cellular assays ?
1.measurement of cellular morphology
2.cell proliferation
3 advantages of animal modals
-inexpensive and easy to handle
-gene knockout strains available
-immunological regents available
disadvantages of in vitro assays
-findings may not always be relevant to tissue/whole organism
disadvantages of in-vivo assays
animals respond differently to infection -diiferent immune and cellular responses
disadvantages of lab research
funding limitations impact use of techniques
some techniques need extensive training
when applying multiple methods to answer the same question they may not give the same result
