Techniques and Omics

Question 1

How are cells cultured?

Answer 1

With nutrient medium containing antibiotic and non essential amino acids (L-Gln)
Foetal calf serum for growth factors
Humidified
5% CO2
Sterile

Question 2

Why are cancer cells cultured?

Answer 2

Easier to isolate
easier to genetically manipulate
limitless replication

Question 3

What are genes inserted with?

Answer 3

Selection cassette
Inducible promoter
Tags

Question 4

What is immunoblotting?

Answer 4

Use of antibodies to bind protein sequence or tag

conjugated enzymes produce visible product

Question 5

Why is immunoblotting used?

Answer 5

Detects presence/concentration (or absence) of protein

With SDS PAGE to detect size

Question 6

How can antibodies be used in live cells?

Answer 6

Fluorescence microscopy with added tags

Question 7

How can fluorescence microscopy be made more sensitive?

Answer 7

Multiple layers of antibodies?

Question 8

How can organelles be fluorescently stained?

Answer 8

Nuclear dyes
Mitotracker
Lysotracker
labelled ligands

Question 9

How can fluorescence be used for cell sorting?

Answer 9

Cell surface receptors detected in flow cytometry

Question 10

How many cells does flow cytometry screen?

Answer 10

Large populations, each cell individually as they past the detector

Question 11

Can can organelles be separated?

Answer 11

Subcellular fractionation

Question 12

What are the 2 types of subcellular fractionation?

Answer 12

Differential centrifugation based on mass

Sucrose density gradients based on buoyancy (lipid:protein)

Question 13

What is the fragment length of parallel sequencing approaches?

Answer 13

50-200bp

Question 14

Why are genomics studied?

Answer 14

diagnoses
treatment 
bioprocessing
agriculture
bioarchaeology

Question 15

Why must 100s of tumour cells be studied for genomics?

Answer 15

Tumours are not heterogenous

Question 16

What are the most common cellular pathways identified by cancer genomics?

Answer 16

Wnt/B-catenin
p53
chromatin remodelling
PI3K/Ras
ER oxidative stress

Question 17

What has cancer genomics shown about the frquency of mutations?

Answer 17

74% mis-sense
14% insertions/deletions
12% splice/nonsense

Question 18

What are metagenomics?

Answer 18

The study of an the average of a populations genome (e.g. human microbiome)

Question 19

What have metagenomics shown about the size of the human microbiome?

Answer 19

124 adults sequenced

160 species

Question 20

Why does transcriptomics vary between cells?

Answer 20

Cell cycle stage
Type of cell
Alternative splicing patterns

Question 21

How are transcriptomes studied?

Answer 21

Using cDNA to hybridise the DNA probe on microarray

Fluorescence produced can measure abundance

Question 22

How can transcriptomics be used to characterise cancers?

Answer 22

Expression ratios
Comparison to standard
Used to give a signature and prognosis

Question 23

How many genes have transcriptomics shown to be implicated in cancer?

Answer 23

67 genes commonly expressed in all tissues

Question 24

How were transcriptomics used to show the effects of salmonella?

Answer 24

Production of IL6, 10 represses production of cytokines, causing gastroenteritis

Question 25

What is the technique used in transcriptomics?

Answer 25

RNAseq with paired end reads

Question 26

How many proteins can be studied in a proteomics sample?

Answer 26

6000

Question 27

What are the proteomic techniques?

Answer 27

Mass spectrometry/ trypsin digestion to give peptide mass fingerprint
Tandem mass spectrometry to give sequence
SILAC

Question 28

Which amino acids does SILAC target?

Answer 28

13C Lysine or Arginine

Question 29

How has the JURKAT T cell line been studied in proteomics?

Answer 29

anti-Fas antibody induces apoptosis by associating mitochondria to nucleus

Question 30

How has proteomics been used to study Dengue Fever?

Answer 30

Difference of 37 proteins following infection