Techniques and Omics Flashcards
How are cells cultured?
With nutrient medium containing antibiotic and non essential amino acids (L-Gln) Foetal calf serum for growth factors Humidified 5% CO2 Sterile
Why are cancer cells cultured?
Easier to isolate
easier to genetically manipulate
limitless replication
What are genes inserted with?
Selection cassette
Inducible promoter
Tags
What is immunoblotting?
Use of antibodies to bind protein sequence or tag
conjugated enzymes produce visible product
Why is immunoblotting used?
Detects presence/concentration (or absence) of protein
With SDS PAGE to detect size
How can antibodies be used in live cells?
Fluorescence microscopy with added tags
How can fluorescence microscopy be made more sensitive?
Multiple layers of antibodies?
How can organelles be fluorescently stained?
Nuclear dyes
Mitotracker
Lysotracker
labelled ligands
How can fluorescence be used for cell sorting?
Cell surface receptors detected in flow cytometry
How many cells does flow cytometry screen?
Large populations, each cell individually as they past the detector
Can can organelles be separated?
Subcellular fractionation
What are the 2 types of subcellular fractionation?
Differential centrifugation based on mass
Sucrose density gradients based on buoyancy (lipid:protein)
What is the fragment length of parallel sequencing approaches?
50-200bp
Why are genomics studied?
diagnoses treatment bioprocessing agriculture bioarchaeology
Why must 100s of tumour cells be studied for genomics?
Tumours are not heterogenous
What are the most common cellular pathways identified by cancer genomics?
Wnt/B-catenin p53 chromatin remodelling PI3K/Ras ER oxidative stress
What has cancer genomics shown about the frquency of mutations?
74% mis-sense
14% insertions/deletions
12% splice/nonsense
What are metagenomics?
The study of an the average of a populations genome (e.g. human microbiome)
What have metagenomics shown about the size of the human microbiome?
124 adults sequenced
160 species
Why does transcriptomics vary between cells?
Cell cycle stage
Type of cell
Alternative splicing patterns
How are transcriptomes studied?
Using cDNA to hybridise the DNA probe on microarray
Fluorescence produced can measure abundance
How can transcriptomics be used to characterise cancers?
Expression ratios
Comparison to standard
Used to give a signature and prognosis
How many genes have transcriptomics shown to be implicated in cancer?
67 genes commonly expressed in all tissues
How were transcriptomics used to show the effects of salmonella?
Production of IL6, 10 represses production of cytokines, causing gastroenteritis
