|qPCR Master Mix||10000+qPCR primers||10000+qPCR standards|
High amplification efficiency
Wide linear range
Relative quantification allows calculation of the ratio between the amount of target gene and a reference gene in a sample. Since this method measures the amount of target gene relative to a presumably invariant control gene, relative qPCR is most often used to measure genetic materials, for instance, between tissues or between diseased and healthy samples. The advantage of this method is that using an internal standard can minimize the variations in sample preparation and handling.
The main applications of relative quantitative analysis by qPCR include:
• Difference analysis of gene expression
• Validation of microarray results
Reference gene and target gene expression analysis by relative quantitative qPCR method
If the amplification efficiencies of the target gene and the reference gene are close to 100%, less than 5%, the Livak method (Ratio=2–∆∆CT) is suitable. If the amplification efficiencies of the target gene and the reference gene are not close, more than 5%, the Pfaffl method (Ratio=(Etarget)∆CT, target (calibrator–test) / (Eref)∆CT, ref (calibrator – test)) is suitable.
Using relative quantification qPCR analyze the gene expression of ICAM1 in HT293 cells.
1. The IL17A protein factor stimulates HT29 cells to express ICAM1 for 24 hours, then the expression analysis of ICAM1 is performed.
2. The antibody for the IL17A protein suppresses HT29 cell2 to express ICAM1 for 24 hours, then the expression analysis of ICAM1 is performed.
Sample names: HT29(control),HT29(+IL17A, 24 h),HT29(+antibody for IL17A, 24h)
Detected genes: SDHA (reference gene) and ICAM1
Detection method: 2–∆∆CT relative quantification (SYBR Green dye method)
Test experiment report:
1. Primer quality verification
2. Report charts of original data for detected samples
Amplification curve of SDHA
Melting curve of SDHA
Amplification curve of ICAM1
Melting curve of ICAM1
3. Analysis report of test results
Analysis results of ICAM1 expression
Graph of different ICAM1 expression