[Quantitative analysis of mRNA level by PCR method using single basemutated template as inner standard]

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Context:

• Quantitative gene expression analysis is crucial for understanding cellular function and disease.
• Accurate quantification of specific DNA or RNA targets is essential in molecular biology research.
• Existing methods may have limitations in sensitivity, specificity, or cost-effectiveness.

Purpose:

• To develop and validate a novel quantitative polymerase chain reaction (PCR) method for precise DNA quantification.
• To introduce a modified DNA template with an introduced restriction enzyme site for internal standardization.
• To apply this method for quantifying the arginine vasopressin V1 receptor (AVP V1 receptor) mRNA in rat liver tissue.

Summary:

• A modified DNA template with an EcoR I restriction site was created using a single base change via PCR.
• This mutant DNA served as an internal standard, co-amplified with the target DNA in a single PCR reaction.
• Following EcoR I digestion and agarose gel electrophoresis, the target DNA copies were quantified based on size separation and band intensity.
• The method successfully quantified approximately 1.25 x 10(-20) mol of AVP V1 receptor cDNA from 1 microgram of total RNA in Sprague Dawley rat liver.

Impact:

• Provides a sensitive and accurate method for quantifying specific DNA sequences, applicable to various research areas.
• Enables precise measurement of gene expression, aiding in the study of physiological and pathological processes.
• Offers a valuable tool for molecular diagnostics and basic research requiring accurate nucleic acid quantification.