Gene expression analysis using Real- time PCR

 

HEREDITY HEALTHCARE AND LIFE SCIENCES

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Dr.Gopal Purohit

Email:gopal.purohit@hdls.in

Contact number:7381298980

Website: www.heredity.in

 

 Written by: Sweta Yadav

 

Introduction:

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. A gene product is the biochemical material, either RNA or protein, resulting from expression of a gene.The products are often proteins.The product is a functional RNA in case of

non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA).

 

 

Overview:

Gene expression and real time analysis was first introduced in 1992 by Higuchi and coworkers and has seen a rapid increase in its use since then (Higuchi et al., 1992, 1993). Over the past decade, real-time PCR applications have become broadly used tools for the quantification of specific sequences in complex mixtures. However, the most common use for this technology has been to study gene expression levels by coupling it with a procedure called reverse transcription. Real-time PCR allows precise quantification of specific nucleic acids in a complex mixture even if the starting amount of material is at a very low concentration.This is accomplished by monitoring the amplification of a target sequence in real-time using fluorescent technology. The time taken for the amplified target to reach a threshold detection level correlates with the amount of starting material present.

 

Real-time PCR —also known as quantitative reverse transcription PCR (RT-qPCR), and quantitative PCR (qPCR)—is one of the most powerful and sensitive gene analysis techniques available. Real-time PCR measures PCR amplification as it occurs, so that it is possible to determine the starting concentration of nucleic acid.

 

Some important questions:-

 

✓ When and where does gene expression occur?

Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule. Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene's DNA is passed to mRNA). Prokaryotic gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the cytoplasm. Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm (translation).

 

 

✓ What controls gene expression?

Gene expression is primarily controlled at the level of transcription, largely as a result of binding of proteins to specific sites on DNA. In 1965 Francois Jacob, Jacques Monod, and Andre Lwoff shared the Nobel prize in medicine for their work supporting the idea that control of enzyme levels in cells is regulated by transcription of DNA. occurs through regulation of transcription, which can be either induced or repressed. These researchers proposed that production of the enzyme is controlled by an "operon," which consists a series of related genes on the chromosome consisting of an operator, a promoter, a regulator gene, and structural genes.

 

✓ What can trigger or prevent gene expression?

Gene expression is influenced by numerous factors, including molecules within the cell, mutations causing dominant negative effects and haploinsufficiency, signaling molecules from surrounding cells and the environment, and epistasis. Various molecules within the cell modulate gene expression.

 

 

Gene Expression Analysis Using Real-Time PCR

 

●    Verification of NGS and Microarray Results using TaqMan Gene Expression Assays.

●    Soil Plant Bacterial/Fungus sybr based gene expression analysis.

●    Immuno-Oncology Research using TaqMan Gene Expression Assays.

●    Complex and Neurological Disease Research using TaqMan Gene Expression Assays.

●    Cancer Research using TaqMan Gene Expression Assays.

●    All virology based Research Using TaqMan Gene Expression Solutions.

●    Immunology Research using TaqMan Gene Expression Assays and Arrays

●    Genetic Variation Analysis Using Real-Time PCR

●    Mutation Detection With Real-Time PCR

●    CFTR Gene Mitation Analysis

●    TaqMan Assays for Disease Research

 

 

Common gene expression research applications:

 

●    Real-time PCR

●    Cancer research

●    Immuno-Oncology

●    Cancer genetics

●    Neurodegenerative and complex disease

●    Validate results from other technologies

●    Pathway analysis

●    Biomarker signatures

 

 

We have developed two chemistries for the sensitive, accurate quantification of mRNA using real-time PCR: Applied Biosystems TaqMan probe based chemistry, and Applied Biosystems SYBR Green dye-based chemistry.

 

 

References:

 

1.  Gibson et al., 1996; Bi'eche et al., 1999; Leutenegger et al., 1999; Livak and Schmittgen, 2001; Liss, 2002).

 

2.  https://www.net/subramaniansethupath/regulation-of-gene-expression-63016286

 

3.  https://sphweb.bumc.bu.edu/otlt/MPH-Modules/PH/DNA-Genetics/DNA-Genetics7.html

 

4.  https://www.slideshare.net/SureshAntre/gene-expression-analysis-by-real-time-pcr-70401928