technique

• Methods to measure Gene Expression/Action of promoters

  1-Reporter assays

  
  

  2- Identify promoter elements using 5' deletion of reporter constructs
• Methods to measure mRNA levels

  1. Northern Blotting and electrophoresis

  
  

  2. RT qpCR (reverse transcriptase quantitative PCR

  
  

  3. Microarrays

  
  

  4. RNA-seq
• Methods to measure Protein levels

  1. Western blot
• Methods to analyse transcription factors-In vivo

  1. Electrophoretic mobility shift assays/ gel shift

  
  

  2. ChIP -chromatin immunoprecipitation
• Methods to analyse transcription factors-In Vitro

  1. Transcription assays

  
  

  2. Reporter assays

  
  

  3. Mutational analysis of activators
• Reporter assays/analysis- investigate promoters

  1. Use promoter element to control expression of reporter gene.

  2. Get transcription of gene and protein produced

  3. Protein level measured-enzyme or fluorescent

  Reporter genes used--Enzymes that produce colour change when substrate present

  -Beta galactosidase/lac Z

  -Luciferase enzyme

  -Fluorescent proteins
• Reporter genes used for reporter analysis of promoters

  Enzymes-

  -Beta galactosidase= X-gal substrate=Blue colonies

  
  

  -Luciferase enzyme=Luciferin substrate=Fluorescence

  Proteins-

  -Green fluroescent protein
• 5' Deletion reporter constructs- Identify promoter elements

  1. Make 5' deletion series of DNA- remove sections from 5' region

  
  

  2. Ligate into plasmid with a reporter gene -transform E.coli and isolate plasmid DNA

  
  

  3. Transfect each plasmid to different cells and identify if reporter gene expressed/what level

  
  

  
  

  =Able to identify which region of the promoter controls the gene expression
• Measure mRNA levels-Northern Blotting

  1. Isolate RNA from cells

  
  

  2. Electrophoresis=separate the RNA based on size

  
  

  3. Might have diff RNA in cell-identify the one of interest

  
  

  4. Transfer the RNA from gel to a membrane

  
  

  5. Hybridise with a radio labelled probe (complementary to a specific mRNA sequence)

  
  

  6. Light up specific band=identifies that RNA when detected via radiography
• Analysing Northern Blot Results-mRNA

  Can expose a cell to two compounds and investigate how this impacts certain mRNA levels.

  
  

  The strength/darkness of band indicates the level of mRNA expressed

  
  

  NEED A LOADING CONTROL-Check that you used the same amount of RNA in each sample
• Loading control in northern blot

  A gene who's RNA levels will not change under the experiment conditions and will be expressed at same level regardless.

  
  

  Check you have loaded the same amount of RNA
• Measure mRNA levels


  1. Isolate RNA

  
  

  2.Hybridise a oligonucleotide DT primer- a primer that can allow DNA replication

  
  

  3. Reverse transcriptase enzyme will produce DNA from RNA (cDNA)

  
  

  4. PCR replicates and amplifies the DNA

  
  

  5. Can then analyse specific genes/RNA levels they are expressed etc
• Measure mRNA using- Micro arrays

  1. use cDNA probes of diff colour red/green and hybridise to ss oligonucleotides complementary to diff to mRNA's (cDNA). Diff one on each spot

  
  

  2. Need control cell to identify changes in mRNA levels- cancer cell vs normal cell encodes a diff colour fluorescent protein e.g green and red .

  
  

  3. Each well represents a diff mRNA type /gene

  
  

  4. If expressed in one -it will be colour of that cell type. if expressed in both- be a mix (yellow)
• Measure mRNA using RNA-seq

  1. Isolate RNA from a sample-enrich mRNA and deplete rRNA

  
  

  2. Fragment

  
  

  3. Convert RNA to cDNA

  
  

  4. Ligate sequencing adapters=amplify

  
  

  5. Next gen seq

  
  

  6. Identify genes in normal vs cancer cell
• Measure protein levels-Western blotting

  1.Separate protein sample via denaturing polyacrylamide gel electrophoresis PAGE

  
  

  2.Transfer to membrane PVDF

  
  

  3. Stain with antibodies specific to diff protein type

  
  

  4. Primary antibody has a secondary antibody that can fluoresce or has enzyme conjugated= Signals level of protein

  
  

  5. Visualise bands

  
  

  NEED lOADING CONTROL-tubulin or actin if comparing diff environments
• Analysis of activators/Transcription factors- Electrophoretic mobility shift assay/gel shift

  IN VITRO- Identify the txn factors (proteins) ability to bind DNA probes

  1. radiolabelled probe DNA and txn factors

  2.Run on non-denaturing acrylamide gel

  3. Free DNA will run quickly down the gel and be further down

  4. DNA bound to txn factor protein=further up the gel

  can mutagenise the probe DNA or protein to identify binding sites
• Gel Shift results- analysing activators

  Tx factor suspected to bind region 50-100bp upstream of start site.

  
  

  Create 3 radiolabelled DNA probes (little sequences)- run on gel with txn factor and identify which band is higher up and so bound

  
  

  Identify shared sequence /overlap of the probes-mst be located here
• Chromatin Immunoprecipitation (ChIP) (IN VIVO)

  Identify binding sites on DNA of activator

  
  

  1. Cross link activator to DNA

  
  

  2. Shear, sonication and isolation of chromatin

  
  

  3. Use antibody specific to activator to extract the DNA with activator bound

  
  

  4. Heat and get rid of cross links, digest the protein

  
  

  5. Use PCR and CHiP Seq to identify binding sites
• Transcription Assays- analyse txn factors/activators IN VITRO

  Need activator with functional DNA binding site and Activation domain

  
  

  -RNA Pol 2

  
  

  -Radiolabelled rNTPs

  
  

  -General TXN factors

  
  

  -DNA template

  
  

  If can bind DNA=get a band of the RNA transcript
• Reporter Assays-analyse txn factors/activators-IN VIVO

  Transfect e.coli with two plasmids

  -One encodes reporter gene with binding site for the activator

  
  

  -One encodes the txn factor

  
  

  =If txn factor works-get expression of reporter gene protein
• Mutational analysis of activators-investigate key domains

  Identify activation domain and DNA binding domain

  
  

  Make truncated proteins with bits missing. Analyse ability to activate txn and ability to bind DNA