Skeletal muscle ‘omics: methods and insights Flashcards
why use omic approaches
what is the hierachy of omic methodologies
- Genomics (gene)
- Transcriptomics (RNA)
- Proteomics (protein)
- metabolomics (metabolites)
what is a disadvantage of WB, RT-PCR etc being reductionist approaches?
- permit examination of VERY limited number of molecules
what does omic analyses allow to be assessed?
allows expression changes in hundreds of thousands of molecules to be assessed
what is are 2 examples of transcriptomics?
- microarrays
- RNA sequencing
microarrays to determine gene expression
outline the process for microarrays to determine gene expression
1) sample mRNA is prepared using protein extraction and RT-PCR and labelled with fluorescent dye
2) specific probes are generated onto microarray
3) sample is added to microarray
4) Specific gene sequence in sample binds with specific probe only
5) Microarray is scanned and fluorescence from sample is detected (more fluorescence intensity = more gene expression)
Transcriptomics: understanding training in younger and older adults
what is one of the purposes of the muscle extra-cellular matrix
- force transduction (generating forceful contractions)
RNA sequencing
outline the process of RNA sequencing for transcriptomics
Extract RNA
↓
Library prep by only selecting poly(A) (the mature RNA) and removing immature RNA,
(Poly(A) only (i.e. mature RNA)
↓
fragment RNA
↓
Add adapters so it adheres to the platform better
↓
Sequence
microarray or RNA sequencing
Compare and contrast the advantages and disadvantages of transcriptomics techniques: microarray and RNA sequencing
microarray:
- £300
- stream-lined, validated with few variations
- well established methods
RNA sequencing:
- expensive
- can take weeks-months to even years, so laborious
proteomics
why is proteomics important
Genes encode proteins, however,
- increased gene expression does not necessarily mean increased protein expression
- Proteins are ultimately the functional units of the cell molecules that regulate metabolism
what are the 3 techniques for proteomics?
- iTRAQ
- MALDI-TOF
- 2D-PAGE
2-D Page
outline, in 5 steps the process of the proteomics technique called 2-D page
- Protein is extracted from sample
- Then subjected to 2D-PAGE by:
- Sample is separated first by charge (pH)
- Then by molecular mass by passing through a gel
- Produces gel with series of spots, with each spot corresponding to a single protein / cluster of structurally similar proteins
- density of spots from two samples then compared
- spots are cut out and analysed by MS to identify proteins
what is the disadvantage of 2D-PAGE?
issues with reproducibility
proteomics - MALDI-TOF
what are 2 advantages of MALDI-TOF
- Allows determination of molecule mass, elemental composition and chemical structure
- TOF has large mass range to cover whole proteome
proteomics
what does MALDI do?
MALDI – fragments sample into small components via UV beam and ionization
outline how MALDI-TOF works in 4 steps
- put spectroCHIP array into MALDI-TOF cylinder
- shoot spectroCHIP with UV light to bombard chip, vaporising and ionising the sample
- when shot with UV light, postively charged sample will fly to the negative detector in nanoseconds, with lighter particles reaching there first, and heavier ones getting there later
- based on the weight of these protein particles on your analysis software, you can identify based on weight what those proteins are
proteomics - iTRAQ
what does iTRAQ stand for?
- isobaric tags for relative and absolute quantitation
outline how iTRAQ works in 5 steps
1) give each sample a unique label and an iTRAQ balancer
2) mix samples into a mixture
3) iTRAQ balancer makes mass all of peptide samples to have the same molecular weight
4) run mixture on a mass spectrometer, which shows all the different samples that you made with peptides at similar peaks due to the differences in balancers added
5) run it through a mass spectrometer again, fragmenting the sample, which removes the iTRAQ balancer, but now all of the peptide samples within the main samples show their actual weights too so you can compare using software analysis
what is iTRAQ combined with and what does this permit?
- combined with MADLI-TOF MS or just MS
what is a downside of iTRAQ
- compared to other proteomic techniques, you only find a limited number of proteins
metabolomics
state what metabolomics measures
measures the plethora of small molecules involved in intermediary metabolism (eg: Fatty acids, sterols, amino acids, sugars, hormones, cytokines)
define a metabolite
any organic molecule detectable in the body with a molecular weight (MW) of < 1500 Da
what are 2 ways you can do metabolomics
- Nuclear magnetic Resonance
- Mass Spectrometry
what is 1 advantage and 2 disadvantage of Mass spectrometry for metabolomics?
advantage:
- highly sensitive so can detect lower abundance metabolites
disadvantages:
- can be expensive
- requires lots of expertise to run
what is 1 advantage and 1 disadvantage of H-NMR?
advantage:
- more affordable than mass spectrometry
disadvantage:
- not as sensitive as mass spectrometry, so can’t detect lower abundance metabolites
