Skeletal muscle omics : methods and insights Flashcards
What is omics?
“ome” and “omics” - suffixes that are derived from genome as a combo of gene and chromosome and genomics (study of genome)
- Genome, Transcriptome, Proteome, Metabolome etc
What is the hierarchy of omic methodologies?
- Gene - Gene sequences (the code) - Genomics
- RNA - The messages - Transcriptomics
- Protein - enzymes/structural proteins - Proteomics
- Metabolites - Small molecules - Metabolomics
Why do we use the omic approaches?
- Top down approach
- Multivariate
- Hypothesis generating
- Model-building to understand complex systems
- Bottom up - approach
- Hypothesis -led
- Uni or bi-variate
- Gene and protein changes are vast, varied and happen acutely and chronicly
Unlikely that a handful of mRNA/protein etc regulate whole organ adaptation
What are the disadvantages of the WB, RT-PCR reductionist approaches?
- Permit examination of a VERY limited number of molecules
What are some benefits of omic approaches?
- Many genes/ proteins may co-vary with muscular adaptation to exercise
- Omic analyses allow expression changes in hundreds - thousands of molecules to be assessed at once
- Provides a global (holistic) approach far beyond what is known of a limited number of molecules
- Might facilitate identification of novel biomarkers . regulating (patho) physiological processes
What is transcriptomics?
- Microarrays (Genechip etc)
-Allows simultaneous measurement of hundreds, thousands or tens of thousands of genes in a single sample
Describe the process of using microarrays to determine gene expression
- Sample mRNA is prepared and labelled with fluorescent dye
- Specific probes are generated onto microarray
- Sample is added to microarray
- Specific gene sequence in sample binds with specific probe only
- Microarray is scanned and fluorescence from sample is detected (more fluorescence = more gene espression)
Describe microarray methodology
- Actual chip size is 1.28cm X4
- millions of DNA strands built up in each location
- Actual strand = 25 base pairs - RNA fragments with florescent tags from sample to be tested
3 Shining a laser light at GeneChip array causes tagged DNA fragments that hybridise to glow
Describe the process of RNA sequencing for transcriptomics
- Extract RNA
- Library prep (Poly A) only i.le. mature RNA
- Add adapters
- Sequence
What is proteomics?
- Similar to genomics, except analyses thee expression of all the proteins in a sample rather than the genes:
Important because: - Increased gene expression does not necessarily mean increased protein expression
- Proteins are ultimately the functional units of the cell molecules that regulate metabolism
- several possible methods for performing proteomics
Describe 2D- PAGE in proteomics
- Protein is extracted from sample
- Then subjected to 2D PAGE
- Sample separated first by charge (pH)
- Then by molecular mass by passing through a gel
- Produces gel with series of spots
- Each spot corresponds to a single protein/cluster of structurally similar proteins
- Density of spots from two samples can then be compared
- Spots are cut our and analysed by MS to identify proteins
- Technique has problems with reproducibility
Describe MALDI-TOF in proteomics
MALDI- Fragments sample into small components via UV beam and ionisation
- Mass spectrometers - glorified weighing machines
- allows determination of molecules mass . elemental composition and chemical structure
- TOF has large mass range to cover whole proteome
What is iTRAQ in proteomics?
- Isobaric tags for relative and absolute quantiation
- Sampled is labelled with a balancer which makes the mass of the samples the same
- Combined with MALDI-TOF ms/ ms, permits quantification of content changes of the proteomes
Wha tis metabolomics?
- measures the myriad of small molecules involved in intermediary metabolism
(FATTY ACIDS, STEROLS, AAS, SUGARSM HORMONES, CYTOKINES) - Resultant signature will provide a profile of how an individual responds to altered activity
What is a metabolite?
Any organic molecule detectable in the body with a MW <1500 Da
