Absolute Quantitation

Absolute Quantitation

Absolute quantitation  assay is used to analyze the change in gene expression in a given sample relative to another reference sample (such as an untreated control sample)

Application 

In various fields of science, including molecular biology, medicine, and environmental research, absolute quantitation is crucial for accurate data analysis and interpretation. Here are a few common techniques used for absolute quantitation:

1. Real-time PCR (qPCR): Quantitative PCR is a widely used method to determine the absolute quantity of a specific DNA or RNA target in a sample. It relies on the amplification of the target molecule using specific primers and a fluorescent probe. By comparing the amplification curve to a standard curve generated with known concentrations of a reference sample, the absolute quantity of the target can be calculated.

2. Digital PCR (dPCR): Digital PCR is a technique that partitions the sample into thousands of individual reactions, each containing a limited number of target molecules or copies. By analyzing the number of positive and negative reactions, the absolute quantity of the target can be estimated. Digital PCR offers higher precision and sensitivity for absolute quantitation compared to qPCR.

3. Mass Spectrometry: Mass spectrometry is a powerful analytical technique used for the absolute quantitation of molecules based on their mass-to-charge ratio. It can be employed to quantify proteins, peptides, metabolites, and other small molecules in a sample. Isotope-labeled internal standards or reference compounds with known concentrations are often used for accurate quantitation.

4. ELISA (Enzyme-Linked Immunosorbent Assay): ELISA is a widely used immunoassay technique that quantifies the concentration of specific proteins or antigens in a sample. It utilizes specific antibodies that bind to the target molecule, followed by detection with an enzyme-labeled secondary antibody. By comparing the signal to a standard curve generated with known concentrations, the absolute quantity of the target can be determined.

5. Flow Cytometry: Flow cytometry is a technique used to quantify and analyze cells or particles in a sample. It measures the fluorescence or light scattering properties of individual cells as they pass through a laser beam. By using fluorescently labeled antibodies or specific dyes, the absolute quantity of specific cell populations or biomarkers can be determined.

These are just a few examples of techniques used for absolute quantitation in different scientific disciplines. The choice of method depends on the nature of the molecule or substance being quantified, the available resources, and the required level of precision and sensitivity.