What is the procedure of expression screening?

Expression screening may be carried out using four main procedures - RNA expression, promoter analysis, protein expression, and post-translational modification. Each of these can be further categorized into specific procedures. 

RNA Expression 

RNA Expression includes 3 different procedures: 

• Northern blotting – In northern blotting, electrophoresis is used to directly quantitate steady-state levels of mRNA, which are then transferred to a membrane where they are incubated with specific problems. Various chemistries or radionuclide labeling is used to detect the RNA-probe complexes. 
• Real-time PCR – A highly accurate and sensitive procedure for expression screening, real-time PCR involves first using reverse transcription of RNA to cDNA to quantitate steady-state levels of mRNA. This is followed by quantitative PCR (qPCR) on the cDNA. The increase in fluorescence signal from DNA-binding dyes or probes is measured during consecutive rounds of enzyme-mediated amplification to determine the amount of each specific target. 
• DNA microarrays – This procedure involves hybridizing labeled cDNA from a sample to complementary sequences on the chip. This enables the visual identification of strongly associated complexes.  

Protein Expression

Protein expression includes three procedures:

• Western blotting – In Western blotting, relative expression levels for specific proteins are quantified by separating extracted cell protein electrophoretically. The cell proteins are then transferred to a membrane and the bound proteins are probed with antibodies that are then detected using various chemistries or radiolabeling. 
• Immunoassays – Immunoassays involve quantitating proteins in solution using antibodies immobilized to a surface or bound to beads that are color coded, followed by detection using a fluorogenic or chromogenic reporter. 
• 2-D gel electrophoresis – In this procedure, a complex mixture of proteins is separated into two dimensions and stained to study differences at whole-proteome level. 

Promoter Analysis

Promoter analysis includes 3 procedures:

• Gel shift assays or electrophoretic mobility shift assays – Gel shift assays are used to study interactions between protein and DNA or protein and RNA, which provides insight into gene regulation. 
• Chromatin immunoprecipitation (ChIP) – ChIP involves chemically cross-linking DNA and protein in living cells, followed by immunoprecipitation of the resulting complex using antibody-coated beads. ChIP enables in vivo identification of the protein-binding regions of DNA.  
• In vitro transcription (nuclear run-on assays) – In this procedure, isolated cell nuclei are incubated with labeled nucleotides and the resultant product is hybridized to a membrane (slot blot). This is then exposed to file or other imaging media to measure transcription rates. 

Posttranslational Modification Analysis

Posttranslational modification analysis two two procedures: 

• Mass spectrometry – Mass spectrometry allows proteins and their modifications to be identified based on their bass. 
• Immunoassays – In immunoassays, specific antibodies are used to detect protein phosphorylation levels and other post-translational modifications.