21: Analysis of gene expression

Question 1

Non-coding RNA-types

Answer 1

tRNA
rRNA
miRNA

Question 2

Northern blot

Answer 2

Method to measure the transcriptional expression of a gene by measuring the level of mRNA directly.

Separation on agarose gel.
Hybridization using a DNA probe specific for the sequence of the gene under investigation.

Question 3

Reporter genes

Answer 3

Gene products easy to assay

Can be used to report on gene expression, detecting the localization of a protein or the presence of a specific segment of DNA, or given if two proteins interact.

Examples:
- Genes for antibiotic resistance
- LacZ gene (encoding β-
galactosidase)

Question 4

Luciferase

Answer 4

Often used as a reporter gene

Emits light when provided with the substrate luciferin.

From lux genes (bacteria) or luc genes (fireflies)

Question 5

Green fluorescent protein (GFP)

Answer 5

• Reporter gene
• Not an enzyme
• Does not require a nonprotein
  cofactor.

Fusion of regulatory regions to the gfp gene => monitor the expression of many genes

Can be used to localize proteins in a cell.
- Binds to C-/N-terminal

Question 6

Gene fusion

Answer 6

Structure in which parts of two genes are fused together, in particular when the regulatory region of one gene is joined to the coding region of a reporter gene.

Question 7

Gel retardation assay

Answer 7

Gel mobility shift assays can be used to determine whether a gene regulatory protein binds to the regulatory region.

Measures change in mobility of DNA during gel electrophoresis. Larger complex runs shorter.

Restriction enzymes are used to cut DNA into segments of 250-1000bp to better observe the difference in size.

Alternatively: use PCR

Question 8

DNA footprinting

Answer 8

Method for testing binding of a protein to DNA by its protection of DNA from chemical degradation.

The DNA fragment is labeled at one end with radioactivity or fluorescence.
Treated with reagent (DNase) that breaks DNA strands unspecific.
Degration of DNA everywhere but in the part bound to protein.
Visible on gel.

The DNA incubated with the DNA binding protein will miss the fragments that were protected against the DNase.
This is visible on the gel as a “footprint” and defines precisely the region in which the protein binds the DNA.

Question 9

Chromatin immunoprecipitation (ChIP)

Answer 9

Technique that identifies the DNA-binding site for a particular transcription factor by crosslinking the DNA to the TF, and then immuno-precipitating the TF.

- Freeze proteins in original 
  locations
- Break down DNA
- Mark POI with specific antigens
- Add magnetic beads with 
  secondary Ab's and centrifuge to 
  isolate the desired complex.
- Sequence the DNA

Question 10

Primer extension

Answer 10

Method to locate the 5’ start site of transcription.
Oligonucleotide primer is bound to mRNA. Reverse transcriptase extends the primer.

Visualized by the incorporation of radioactive/fluorescent tags by rev. tr.

Run on gel to determine the nt where the mRNA starts.

Question 11

Transcriptome analysis

Answer 11

Transcriptome - all the RNA (coding and non-coding) expressed at a given time in a cell.

It is often used to compare how transcript levels change in cells by various treatments, various
conditions, how the transcript levels change over time (time-series), or how they change between different tissue types.

Main techniques:
- RNA-seq (high throughput cDNA
sequencing)
- Microarrays

Question 12

RNA seq

Answer 12

The use of high-throughput cDNA sequencing to characterize an RNA sample.

Necessary to remove rRNA.

Can identify expressed single nucleotide polymorphisms (SNPs) and post-transcriptional editing of mRNA.

Question 13

DNA microarray

Answer 13

Chip carrying array of DNA segments used to simultaneously detect and identify many short RNA or DNA fragments by hybridization.

- DNA printed into nylon- 
  membrane or a glass slide.
- mRNA extracted from cells and 
  labeled
- Transferred to chip
- Intensity of label correlates to the 
  amount of that particular mRNA.

Can compare two different conditions at the same time if the different mRNAs are labeled differently.

cDNA array:
- cDNA generated by PCR
amplification.

Oligonucleotide array:
- ss DNA 20-25 nt long
- Used to simultaneously detect 
  and identify many short RNA/DNA 
  fragments by hybridization. 
- Reactive groups are linked to a 
  glass chip and blocked.
- Each of the four nt are added in 
  turn.
- A mask covers the areas that 
  should not be activated during 
  any particular reaction.
- Light activates all the groups not 
  covered with the mask, and a nt is 
  added. 
- The cycle is repeated.

Question 14

TaqMan quantitative PCR to assay gene expression

Answer 14

Monitoring of the amount of the PCR product in real time.
Can determine expression of multiple genes at the same time.
Has additional probe in addition to the two primers of PCR.

Question 15

Serial Analysis of Gene Expression

SAGE

Answer 15

Method to monitor level of multiple mRNA molecules by sequencing a DNA concatemer that contains many serially-linked sequence tags derived from the mRNAs.

Steps:
1. Extract the mRNA from a cell and 
   convert it to cDNA using an 
   oligo(dT) primer (attached to 
   biotin) that hybridizes to 
   the polyA tail of mRNA.

2. Bind biotin to streptavidin.
3. Cleavage by restriction enz.
   => sticky ends
4. Bind fragments to streptavidin-
   coated magnetic beads.
5. Divide into two samples. Ligate
   to different artificial linkers for
   restriction enz => cut
6. PCR using one primer that binds
   to each of the linkers.
7. Cleavage => sticky ends
8. Ligation => DNA concatemer.
9. Clone + sequence DNA
   concatemer.

Counting and identification of tags indicate the relative levels of the original mRNA molecules.