相对定量（两个样本中的基因表达水平进行比较 ）
Determines changes in steady state transcription of a gene or genes
Expression of the gene/s of interest is normalized against a reference gene/s (housekeeping gene/s) with no or insignificant expression variation
Examples of some reference genes/housekeeping genes used : b-Actin, GAPDH, 18s rRNA
b-2 Micro globulin, Cyclophilins
溶解曲线（Melt Curve ）
Only possible with DNA Binding dyes (SYBR Green I) and after completed real time PCR
Determines the temperature at which 50% of the DNA molecules separate into two strands - or “melts” apart
Discriminates by Melting Temperature (Tm), Tm is dependent on:
- sequence (G/C content)
- length
Fluorescence vs. Temperature 
The second figure is the raw data. We are recording fluorescence vs temperature. We see a slow decrease in fluorescence that is non-specific. We then see an inflection point. This rapid decrease in fluorescence is due to the melting of double stranded DNA. As this DNA disassociates the SYBR green is released and no longer fluoresces. The center of the inflection point is the Tm, the point at which half of the DNA has disassociated.
The third figure is the negative 1st derivative of the second graph. This allows us to better visualize inflection points that may be on top of each other.
The center of the peak is the Tm of the PCR product and each peak represents an individual product.
溶解曲线检测引物特异性
Melt curve showing two amplified products
You can use melt curve to check the specificity of your reaction. Here you can see clearly that more than one product is being made - you should check the specificity of your primer and target sequences in BLAST and also run it on a gel.
