Why do the same primer real time PCR amplification efficiency is not the same every time?
Efficiency is influenced by the freshness of the reagents (freezing and thawing decreases primer efficiency), your pipetting skills, any errors in measurements, air bubbles, etc. If you see a large difference in efficiency, it's most likely a pipetting error and/or your primers or enzyme mix are too old.
The main factor influencing the amplification is the cDNA, which is a reflection of your RNA preparation. Of course every RNA extraction has a different chemical purity and integrity (you should check them with a NanoDrop and a gel or a BioAnalyzer run, respectively, every time, to have an idea of how consistent is your RNA quality). Consequently the cDNA quality will be different. A slight variation you will also have producing different cDNAs from the same RNA, depending on how clean was the RNA to start with and how you preserve it. You will increase consistency keeping the RNA in the 70% ethanolic solution you precipitate it in, and spinning every time a fresh aliquot.
Within the same cDNA, it will help inactivating the RT at the end of your reverse transcription by heating at 85 °C for 10 min, because the RT in lack of dNTPs activates its reverse function of 5'-3' exonuclease and will destroy the cDNA. Of course thawing and freezing the large cDNA molecules also affects their integrity, this is unavoidable, but you can aliquot your cDNA to reduce the effect.
Keep in mind that the standard curve slope is quite constant within the same RNA preparation, even if you lose template material and therefore increase the Ct, because it depends on the chemical purity and not on the amount of starting material. So it will not change slope, although it will move upwards, to higher Cts. But you have to be careful not to end up outside of the linear range of your calibration curve if your starting material drops too low (but this you will know from the standard curve itself, there is a Ct value on the higher side after which you cannot connect anymore the Cts with a straight line to the previous ones... and the same happens at low Cts, where the starting template is too much!).
Pipetting errors are usually little, unless you are making gross mistakes. You can check your accuracy in the pipetting looking at the Cts of the technical replicates (you should have normally 3): the variation (SEM) within them should be less than 0.5%, you'll see that often is below 0.2%.
- Badges
- Science method