Ways to assess gene expression

in situ hybridization

• mRNA localization in an organism

• Fixed cells  

• Probe binds RNA 

• To help study a pathway at the RNA level, shows when RNAs are expressed, in what cells they are expressed, and how long the RNA stays in cells

• Determine which cells are producing a secreted protein

• Use fluorescent in situ hybridization to measure transcriptional activity of a pathway 

fluorescent in situ hybridization

measures transcriptional activity of a pathway 

immunofluorescence

• Protein localization  

• Fixed cells  

• Probe binds RNA 

RT-PCR (Reverse Transcription Polymerase Chain Reaction) 

measures level of mRNA

Western Blot

measures amount of protein

Reporter genes

• Looks at promoter activity and allows you to see which cells are expressing certain genes 

• living or fixed tissues

• Cells that would be expressing the gene will now be expressing GFP

• reports when and where transcription of the gene happens

• When you want to track a certain “type” of  cell

• When you want to determine gene expression over time 

• tracks dynamics of upstream signaling

fusion proteins

• look at protein activity

• gene + GFP

• living or fixed tissues

• When you want to track a specific protein over time

• When you want to determine localization of a specific protein