CMB2000/L20 High-Throughput Research Flashcards
How long has the shift to high-throughput approaches taken?
20-25 years
Why has the transition to high-throughput research been so slow? (2)
Skills gap - lack of training
Messiness - noisy and unpredictable data
Give 2 approaches used before high-throughput research became prevalent.
Formaldehyde gel electrophoresis
DEPC treated everything
Radiolabelled RNA probes
Name 2 new technologies made possible by high-throughput research.
High throughput sequencing
Mass spectrometry
Cytometry
Imaging
What can be analysed by high throughput sequencing? (3)
Genomes
Transcriptomes
Epigenomes
What can be analysed by mass spectrometry? (3)
Proteomes
Metabolomes
Lipidomes
Give 3 scales in high-throughput experiments.
Number of parameters measured
Number of observations made
Number of biological replicates
What do mass spectrometers consist of? (3)
Ion source
Mass analyser
Detector system
Define mass spectrometry.
Analytical technique that measures mass:charge ratio (m/z)
Explain mass spectrometry using trypsin as an example. (4)
Proteases cut at defined sites
Trypsin cute C-terminal of K or R
Proteins cut with an enzyme will give series of peptides of different masses
This is peptide mass fingerprint
Define cytometry.
Technique for quantitatively measuring markers on cells
Explain cytometry. (3)
Labelled antibody binds to marker and is detected
Cells can be retained (cell sorting)
Historically low-throughput (small number of markers)
Describe dimensional flow cytometry.
Modern fluorescence flow cytometer
Analyse multiple features per-cell
Rapid analysis of 100,000s of cells
Describe mass cytometry. (4)
Remove dependence on fluorescence
Detect antibodies by heavy metal tags (time of flight mass spec)
Up to 40 parameters
No spectral overlap
Cells destroyed
Give 3 difficulties with high throughput.
Noise can drown out signal
Prone to experimental effects - increases noise
Expensive - tendency to reduce sample size
