When SYBR® Green dye is used in real-time PCR, any primer dimers will cause higher background, and may lead to a generation of CT<40 for NTC samples. The primer dimer is not usually detected unless a dissociation profile is generated. Because the background is higher, large amounts of primer dimers may alter the CT of your experimental samples and change the expression level interpretation.
A SYBR® Green amplification plot prior to the dissociation curve. In this view the presence of primer dimers cannot be determined.
|
A dissociation curve following a SYBR® Green real-time reaction. The presence of primer dimers can be easily identified in the dissociation curve by the additional peak(s). The primer dimer peaks are usually seen at low melting temperatures. |
Solution
Optimize primer concentration
using the following combinations:
|
Reverse Primer (nM) |
Forward Primer (nM) |
||
|
50 |
300 |
900 | |
|
50 |
50/50 |
300/50 |
900/50 |
|
300 |
50/300 |
300/300 |
900/300 |
|
900 |
50/900 |
300/900 |
900/900 |
Use an amount of primer that produces no primer dimer and gives optimal amplification efficiency.