Lecture 14 - Gene Expression

Question 1

how does gene expression differ

Answer 1

• by region in multicellular organisms
• in development
• in dynamic response to environmental signals
• in disease states
• by gene activity

Question 2

what is the target of gene expression studies

Answer 2

mRNA

Question 3

what type of RNA is most abundant in cells

Answer 3

rRNA, >70%

Question 4

what type of RNA is least abundant in cells

Answer 4

mRNA, 3-4%

Question 5

how is gene regulation mediated through RNA

Answer 5

• transcription
• RNA processing
• RNA export
• RNA surveillance
• siRNA

Question 6

what does Northern Blotting detect

Answer 6

specific RNAs

Question 7

how does Northern Blotting deal with RNA

Answer 7

it isolates RNA from cells, separates them using electrophoresis, and probes them with labeled cDNA from a specific gene

Question 8

how is RNA quantified in Northern Blotting

Answer 8

signal intensity

Question 9

what are the steps in creating cDNA libraries

Answer 9

• isolate RNA
• convert RNA to complementary DNA through reverse transcriptase
• subclone the RNA into a vector
• transform and select for it in E. coli
• sequence the cDNA inserts

Question 10

what are microarrays

Answer 10

solid supports such as a membrane or glass microscope slide on which DNA of known sequence is deposited in a grid-like array

Question 11

how are microarrays used to measure gene expression

Answer 11

• RNA is isolated from matched samples of interest
• RNA is converted to cDNA
• it is labeled with fluorescence or radioactivity
• it hybridizes to microarrays to measure the expression levels of thousands of genes

Question 12

how do two colour microarrays work

Answer 12

• you have a test and reference sample
• each one is assigned a colour
• the colours on the microarray indicate which cells express those genes
  e.g. red -> present in pathological cell, green -> present in normal cells, yellow -> present in both cells

Question 13

what is the brightness proportional to in a microarray

Answer 13

amount of cDNA bound to the spot on a chip

Question 14

what are the steps of RNA sequencing

Answer 14

• RNA is isolated from the target cells/organisms
• RNA is converted into cDNA
• high-throughput sequencing is used to sequence reads (e.g. using Illumina)
• reads are mapped to genes/genomes and reads are counted

Question 15

what does data normalization do

Answer 15

separates true variation from variation due to experiment variability

Question 16

what is an example of data normalization

Answer 16

using relative changes rather than absolute changes; fluorescence can be calculated as a ratio between experimental/control groups