Question

What three methodologies were used to assess gene expression? Give a brief description of each methodology (the principle of the methodology, not a summary of the actual experiment performed). What is/are the primary advantage(s) of each method? (15 marks)

Answer 1

The four methodologies to study gene expression are as follows

1- Northern Blot/ SAGE

2- RT-PCR

3- Western blot

Northern Blot / SAGE

Both help to identify and analyse gene expression, which genes are turned on and off in a cell. This further helps to study what triggers gene expression. They measure the levels of mRNA. The cell needs to copy the DNA sequence of gene into mRNA transcript to activate gene. By deterring mRNA one can determine which genes are expressed in cells.

Northern blot is used to quantify the mRNA transcript for a single gene. It directly reflects how much transcription of gene has occurred and how vigorously the gene is expressed.

Advantage- used to primarily quantify mRNA and identify quantity of mRNA expressed by two different cells at different times.

SAGE identifies and counts the mRNA transcript in a cell with the help of genetic code called tags. this tag helps match mRNA transcripts with specific gene. All tag contains unique information to identify a mRNA transcript. since the tag read in a continuous sequence therefore it is called serial analysis of gene expression.

Advantages- it measures expression of both known and unknown gene. it is used to discover new gene.

RT-PCR- It is most powerful and sensitive gene expression analysis method. It is fast and convenient. It works by combining RNA template to DNA template using reverse transcriptase enzyme. PCR is used to amplify specific DNA target. cDNA is used as a template for amplification. The monitoring and amplification of reaction is carried out using fluorescence resonance energy transfer ( FRET).

Advantages- analysis of gene expression, genotyping, biomarker discovery, measuring quantity of RNA.

Western blotting- it is also called as immunoblotting or immuno-chromatography. It involves transfer of biological samples from gel to membrane and detect them on surface of membrane. It detects protein based on molecular weight.it involves the principle of SDS-PAGE. The separated protein is transferred to nitrocellulose membrane and are detected using specific primary antibody and secondary enzyme labelled antibody substrate. finally the bands are observed after autoradiography.

Advantages- identification and analysis of proteins based on molecular weight.