omics

Question 1

RNA-seq

Answer 1

sequencing of isolated RNA molecules by 2nd generation sequencing method. You sequence in one run billions of molecules. The sequenced fragments are aligned on the genome. By counting the amount of sequenced RNA fragments per gene you know the gene expression.

Question 2

EST

Answer 2

Expressed sequence tags
take mRNA from a certain tissue and then clone these RNA molecules in a vector. You now
have ESTs. These ESTs are then sequenced by Sanger sequencing. Gene expression levels can now be determined by counting the number of ESTs you find per gene

Question 3

difference between northern blot and microarray

Answer 3

northern blot: only 1 gene
With a northern blot you use a labelled gene specific probe (most of the times DNA)
to detect the presence of an RNA molecule on the blot
microarray: use a labelled RNA or cDNA sample
to detect to which gene specific probes on the array it can bind. These probes present on the array can either be complete or partial cDNAs or oligos

Question 3

cDNA microarray

Answer 3

used to measure competitive hybridisation
o Overexpression of one gene
o One wins the competition -> colour shown

Question 4

oligonucleotide microarray

Answer 4

only hybridise one labelled sample to the probes on the array. used to compare expression level of genes

Question 4

biological variability

Answer 4

variability within the biological sample

Question 5

technical variability

Answer 5

changes during experiment that is indicated

Question 6

difference between loop design and reference design

Answer 6

loop: compare with samples before and after ( comparison forms a loop)
reference: compare with 1 sample used a reference

Question 7

what could be the challenge in proteomics experiments?

Answer 7

• Proteins are much more difficult to characterize and work with than nucleic acids.
• Proteins have 20 building blocks, highly variable structure, hydrophobicity, and charge
• Proteins need to be kept in a functional correctly folded state for most proteomics application

Question 8

5 steps of peptide mass fingerprinting

Answer 8

1. fragment protein into peptides
2. place charges on peptides + vacuum
3. acceleration on magnetic field
4. separate fragments + detection
5. compute m/z ratio and identify protein

Question 9

difference between hard and soft ionization

Answer 9

Hard ionization: ionization occurs due to interaction with energetic electron
Soft ionization: ionization occurs due to interactions within the gas phase

Question 10

What is the difficulty with measuring metabolites?

Answer 10

• Chemical complexity
• Dynamic range
• Dynamic flux
• Non-linear with annotated gene and protein databases